In 1502 Leonardo entered the service of Cesare Borgia, duke of Romagna and son and chief general of Pope Alexander VI. In his capacity as the duke's chief architect and engineer, Leonardo supervised work on the fortresses of the papal territories in central Italy. In 1503 he was a member of a commission of artists who were to decide on the proper location for the David (1501-1504, Accademia, Florence), the famous colossal marble statue by the Italian sculptor Michelangelo, and he also served as an engineer in the war against Pisa. Toward the end of the year Leonardo began to design a decoration for the great hall of the Palazzo Vecchio. The subject was the Battle of Anghiari, a Florentine victory in its war with Pisa. He made many drawings for the decoration and completed a full-size cartoon, or sketch, in 1505, but he never finished the wall painting. The cartoon itself was destroyed in the 17th century, and the composition survives only in copies, of which the most famous is the one by the Flemish painter Peter Paul Rubens (1615?, Louvre).

During this second Florentine period, Leonardo painted several portraits, but the only one that survives is the famous Mona Lisa (1503-1506, Louvre). One of the most celebrated portraits ever painted, it is also known as La Gioconda, after the presumed name of the woman's husband. Leonardo seems to have had a special affection for the picture, for he took it with him on all of his subsequent travels.

Although Leonardo produced a relatively small number of paintings, many of which remained unfinished, he was nevertheless an extraordinarily innovative and influential artist. During his early years, his style closely paralleled that of Verrocchio, but he gradually moved away from his teacher's stiff, tight, and somewhat rigid treatment of figures to develop a more evocative and atmospheric handling of composition. The early painting The Adoration of the Magi introduced a new approach to composition, in which the main figures are grouped in the foreground, while the background consists of distant views of imaginary ruins and battle scenes.

Leonardo's stylistic innovations are even more apparent in The Last Supper, in which he represented a traditional theme in an entirely new way. Instead of showing the 12 apostles as individual figures, he grouped them in dynamic compositional units of three, framing the figure of Christ, who is isolated in the center of the picture. Seated before a pale distant landscape seen through a rectangular opening in the wall, Christwho has just announced that one of those present will betray himrepresents a calm nucleus while the others respond with animated gestures. In the monumentality of the scene and the weightiness of the figures, Leonardo reintroduced a style pioneered more than a generation earlier by Masaccio, the father of Florentine painting. A 22-year project to remove accumulated dust and grease as well as earlier repainting from the mural was completed in 1999.