Bronzino depicts Saint John the Baptist's muscular figure in a restricted space, his torso twisted to accommodate the panel’s vertical format which creates a series of strong diagonal accents, zigzagging the length of the painting. This artificial, serpentine pose marks Bronzino’s move away from naturalism toward a more abstract elongation of forms and more stylized figures, a shift which occurred in the 1540s.

The long, vertical format of this work corresponds to its original function as part of a triptych altarpiece in the private chapel of Eleonora di Toledo in the Palazzo Vecchio, Florence. Bronzino decorated the chapel between 1540 and 1565, as a celebration of the Medici dynasty. The larger, central panel of the original altarpiece (now in the Musée des Beaux-Arts et d’Archéologie, Besançon, France) depicted the Lamentation, which was flanked by the present painting on the left, and Saint Cosmas on the right (a fragment of which is today in a private collection). The saints were chosen by Bronzino for their relevance to the Medici—Saint John was the patron saint of Florence, while Saint Cosmas was the name saint of Eleonora's husband, Cosimo de' Medici.

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