Leonardo was interested in Arezzo both for its central location in the system of roadways and its strategic importance. The city, of very ancient origin, possesses unique archaeological testimonials, such as the Roman amphitheatre, encapsulated in the Monastery of San Bernardo and today included in the itinerary of the Gaio Cilnio Mecenate National Archaeological Museum. The archaeological rediscovery of Arezzo took place quite early, starting in the mid-sixteenth century, when the construction of the Fortress brought to light exceptional finds from Etruscan times such as the Chimera, a bronze statue from the 5th-4th century B.C. that was restored by Benvenuto Cellini. The Archaeological Museum contains Hellenic, Etruscan and Roman finds coming from Arezzo and its territory that influenced Donatello, Verrocchio and Leonardo himself. Displayed in the State Museum of Medieval and Modern Art is a marble putto (puer mingens) by Leonardo's nephew, Pierino Da Vinci.

The ancient Etruscan city of Artimino was a reference point, both strategically and within the system of roadways, for Leonardo's Valley of the Arno and Montalbano. Bearing witness to the existence of the city, destroyed in Roman times, were the remains of walls and emerging archaeological finds. Some of these were walled into the structure of the Romanesque Pieve of San Leonardo. Other testimony is found, instead, in the Archaeological Museum set up within the walls of the medieval stronghold. Here a section is dedicated to the ceramics of Bacchereto, the town where Leonardo's family owned a kiln "da orioli" and where he presumably had his first artistic experience.

In the vicinity of Artimino is the Golfolina, mentioned by Leonardo in the Codex Leicester (ff. 8B-8v, 9A-9r), held by popular tradition to be an Etruscan "cut". Even if this hypothesis is not true, we must remember that on the hills of Grumaggio, just opposite the Masso delle Fate and above the stone quarry, the necropolis of Prato Rosello and the Etruscan city of Artimino stretched out.

According to another tradition, Golfolina, defined by Leonardo as «stone in ancient times joined with Monte Albano in the form of a very high barrier», was "cut" by Ercole Arameo, descendent of Noah and father of Thuscus, for whom the region was named. This myth was reported in 1497 by Giovanni Nanni (Annio da Viterbo) in his Antiquitatum variarum libri, in which the author stated that he had taken this information from the texts of Beroso the Caldean.

Outstanding among the rare reproductions of ancient buildings found in Leonardo's drawings is one of a hypogean tumulus with several chambers, which is today thought to be the funerary complex brought to light on January 29, 1507 in the vicinity of Castellina in Chianti. This drawing (Louvre, Cabinet des Dessins), datable to those years, represents in fact a structure with ceilings built of projecting slabs in the form of a false vault similar to that of the monument from the 4th century B.C. at Castellina. Other details, however, show that the drawing, in the past attributed to Francesco di Giorgio Martini and long thought to be inspired by an Etruscan tomb at Cerveteri, was not intended as a scientific representation of the funerary building. Instead, Leonardo used this structure as a basis for conducting his research on universal forms, which "have in themselves [as Palladio thought for the Pantheon] the figure of the world". The cruciform six-ray "wheel" at the base of the temple designed by Leonardo seems, in fact, to constitute a "multiplication" of the ground plan of the largest tomb on Monte Calvario, which has a dromos partially open and partially roofed with slabs, a vestibule with two side cells and a large chamber at the back. In all, the circular tumulus contains four tombs in coincidence with the four cardinal points. The style of the drawing recalls, moreover, in the treatment of the section and elevation, the drawing of a fortress on f. 117r of the Codex Atlanticus (c. 1507).

On map RLW 12278 Leonardo very clearly evidences the city of Chiusi, a centre of primary importance since Etruscan times, as shown by its numerous treasures now in the local National Museum and the Florence Archaeological Museum. The decline of the city, beginning in the Middle Ages, was caused by the swamps that progressively invaded the Val di Chiana. Chiusi is recalled by Leonardo, in the early 16th century, in Ms. L (f. 94v) in which he describes the itinerary from Buonconvento to Chiusi and from there to Foligno. The rediscovery of the Etruscan civilization also exerted its influence on Antonio da Sangallo the Younger and Leonardo, as exemplified for the latter by the study on the mythological tomb of King Porsenna found in the Codex Atlanticus.

Leonardo showed his interest in the Etruscan city of Cortona on maps RLW 12278 and RLW 12277. He also mentions it twice on map RLW 12682 for the measurement of distances: «from Castiglione to Cortona 5 miles» and «From Foiano to Cortona 8 miles ».

In the Etruscan Academy Museum is displayed the Muse Polymnia, a painting on slate once considered a Roman work, today deemed a Renaissance falsification, perhaps related to Leonardo's rediscovery of the "encausto " pictorial technique, or, according to others, an eighteenth-century work inspired by the coeval Pompeian discoveries. In the Etruscan Library is a seventeenth-century copy of the Book on Painting, (Ms Zuccari, 297): "Opinion of Leonardo da Vinci. Method of painting perspectives, shadows, distances, heights, depths from close up, from afar, and other things".

Of this Etruscan-Roman city, Leonardo may have known the walls (extending for a perimeter of around 2.5 km), a feasible hypothesis considering that, in 1503, he bought two small lots of farmland with olive trees, fruit trees and a stone quarry, located close to the Oratory of Sant’Apollinare and the ancient walls. It is highly improbable instead that he knew about the ruins of the theatre and the temple.

Leonardo was interested in the "Fiesole Lake", which in ancient times covered the Florence-Prato-Pistoia plain; he drew the hills of Fiesole on folio 20v of Madrid Ms. II.

In the "Codex on the Flight of Birds" Fiesole is mentioned three times: once for the sighting of a "bird of prey" on March 14, 1505 or 1506, twice for his prophecy on flight from Monte Ceceri, and once in Ms. G, in regard to the "profile of a stone slab from Fiesole".

On map RLW 12278, Leonardo indicates this ancient town south of Siena. In its vicinity he indicates "Munistero", probably to be identified as Vescovado di Murlo. Toward the end of the 7th century B.C., not far from the medieval centre, in the Poggio Civitate locality, there stood an imposing Etruscan palace, brought to light in the second half of the 20th century.

In Ms. L Leonardo indicates twice, around 1502, the coastline and promontory of Populonia, the only great Etruscan city founded on the sea, with its port in the Gulf of Baratti between Piombino and San Vincenzo. Populonia reached its height of splendour in the 7th and 6th centuries B.C, although already in the 8th century the systematic exploitation of the metal mines on the nearby Island of Elba had begun. Testifying to this past splendour are the grandiose architecture of the tumuli, the funerary trappings and the archaeological finds from ancient ships wrecked in the Bay of Baratti.

Under the ancient name of "Suana", Leonardo represents with great emphasis the Etruscan city of Sovana, in the Commune of Sorano, on maps RLW 12278 and RLW 12277.

It had been an important city since the 8th century B.C., retaining its central position in Imperial times as well. The Etruscan remains are noteworthy: streets such as Il Cavone, the rupestrian necropolis, the tombs of Hildebrand and of the Siren. The Rocca Aldobrandesca is what remains today of the ancient medieval stronghold.

Leonardo draws the Etruscan city of Volterra with many details, from two different viewing points, on maps RLW 12683 (from the east) and RLW 12278 (from the west) and notes its strategic importance in relation to the Val di Cecina and the Valdera on the hydrographical map RLW 12277.

In Medieval and Renaissance times the territory of Volterra was the target of the expansionist policies of Pisa and Florence, because of its mining resources. Undoubtedly, Leonardo knew the architectural remains of the Etruscan-Roman city, such as the walls, which in the 4th century B.C. measured 7,280 meters, and the gate called Porta dell’Arco.

The refinement of the arts and the wealth of ancient Volterra are amply demonstrated by the finds that, since the early 18th century, have been attentively safeguarded in the Guarnacci Museum.