During Emperor Charles V's stay with Isabella of Portugal at the Royal House of the Alhambra in Granada in 1526, it was decided to undertake various rebuilding projects in this sector of the Nasrid palaces, which had already been refurbished in part by Ferdinand and Isabella in order to adapt them to the new Castilian customs. The rebuilding, accomplished between 1528 and 1539, involved the application of a new scale and formal design, with elements alien to Islamic tradition such as fireplaces, decorative features drawn from classical repertoires, and coffered ceilings with Renaissance-style ornamentation. An example is the so-called Emperor's Bureau, which was refurbished in 1532 by Pedro de Machuca, responsible for the design of the Palace of Charles V. The walls must have been decorated with rich tapestries that have not been preserved. The rest of the imperial apartments, designed to house the monarchs' bedchamber and rooms for private rest, were arranged around the Lindaraja garden. The first room is dominated by a great fireplace whose frontispiece is crowned by the imperial coat-of-arms with the recognisable figure of the two-headed eagle upon a globe, a symbol of the spread of the dominions of the Spanish and Portuguese Empire across the Old and New Worlds. Noteworthy too is the ceiling of hexagonal coffers with the initials K and Y (for Charles and Isabella). The next two rooms, known as the "Halls of the Fruits", contain one of the most outstanding iconographic programmes of the Spanish Renaissance. The ceilings were painted by Julio Aquiles and Alejandro Mayner, pupils of Raphael Sanzio and Giovanni da Udine, in about 1537. These works may be considered the earliest set of still lifes to be found in Spain, alternating with the anagrams K and I (believed by some to be the initials of Charles and Isabella, while other specialists see them as referring to the Emperor alone in his attribute of 'KAROLUS IMPERATOR'). Their peculiarity is that they incorporate as motifs the species of fruit from the orchards of the Generalife, and they are stylistically linked with those found in other residences of Italian princes contemporary with these rooms. Regrettably, the Emperor and Empress were never able to occupy these rooms, since the historical and political events which took place after their stay in Granada were to prevent them from doing so.

The Queen's Closet, so called because it was later put to this use, originally had a completely different function. The Nasrid tower of Abu-l-Hayyay (1333-1354), adjacent to the passage along the wall and originally meant as a privileged vantage point for the sultan's exclusive use with views over the surrounding countryside, was converted into a studiolo or stuffeta for the palace's new owner. The space is shared by these two very different cultural traditions, with its original use as a belvedere maintained and, in this case, enriched by an excellent ornamental programme worthy of the greatest Renaissance princes. The stuffeta was a resource employed in the noble residences of Italian princes. It consisted of a large slab of pierced marble through which aromatic vapours entered from a water boiler underneath, producing a luxurious ambience propitious for retirement and meditation.

The frescoes, painted by Aquiles and Mayner between 1539 and 1546, represent mythological scenes like the "Fall of Phaeton" or scenes from Ovid's Metamorphoses, recalling the Vatican Stanze with their motifs inspired by those of Nero's Domus Aurea. Also included are contemporary scenes pertaining to the Emperor's biography such as the Conquest of Tunis, an event which occurred in 1535 with a vast deployment of the imperial fleet. The scenes are based on sketches by the Dutch painter Cornelisz Vermeyen, who accompanied the Emperor himself during the action, and they also show the ports of Cagliari, Sicily and Tripani and the ruins of Carthage. The painted pilasters are decorated with motifs proper to the formal vocabulary of Italian classicism alongside others of a more curious nature, including in particular one of the first pictorial representations of corn cobs, while next to them is the two-headed eagle, the symbol of the Emperor, to identify the person for whom the room was destined. The Queen's Closet is in fact a studiolo designed for a true Renaissance prince incarnated in the figure of Charles V. Not only is it a conception for the new Royal House, but it also incorporates, in the very heart of the Islamic palaces, a set of rooms that express the refinement and taste of the culture of Renaissance classicism, also a symbol of the cultural synthesis represented today by the monumental complex of the Alhambra and the Generalife.