Rosales depicts the moment when the Catholic Queen dictates her last will and testament at Medina del Campo on 12 October 1504, just days before she died. Isabel stands out in a royal chamber almost in darkness, in a canopy bed, with a medal of the Order of Saint James on her bosom. The white sheets contrast with the muted colors of the other personages. The only note of color is the red robe of a grieving King Fernando, whose daughter, Juana, is at his side. Cisneros, in his Cardinal's clothing, is recognizable among the other figures.

This work was painted by Rosales during his stay in Rome. Following a profound reflection as to the best historical subject to depict, he made one of the finest historical works in all of Spanish painting. It recalls the atmospheric realism of Velasquez's works, giving the setting a rather vaporous aspect. The confident lines, the quality of the textures, and the perfect composition —with the brightly lit queen as the work's main point— show the degree of pictorial commitment attained by Rosales in this work.

It was acquired on 22 February 1865 and later moved to the Museum of Modern Art.