The Medicean Theatre, designed by the architect Bernardo Buontalenti, was inaugurated in 1586. Rebuilt only three years later, it presented a U-shaped hall with tiers of seats, the typical conformation of the first Baroque theatres, and an innovative technical feature that consisted of a floor sloping slightly downward toward the stage, designed to provide all of the spectators ith a good view. According to the concepts of the time, in which modern reflections on the art of perspective were applied, the vanishing lines of the scene converged toward a single point, which coincided with the prince's box.

The hall was equipped with complex theatrical apparatus consisting of rotating periaktoi (large triangular prisms used already in Graeco-Roman set design) and sliding backdrops that, along with other ingenious devices, such as big trapdoors through which machines appeared from below, and panels manoeuvred from the ceiling, were utilised for special effects and for changing scenes.

Of the theatre, which originally occupied the first and second floor of the Uffizi in height, there remains today only the Vestibule, onto which open the three doors of the foyer, and the entrance door to the theatre, now occupied by the Cabinet of Prints and Drawings.