Though it was first opened to the public on July 24, 1838 to celebrate Queen regent Maria Christina's name-day, it was soon closed and reopened on May 2, 1842 by the twelve-year-old Queen Isabella II of Spain who had an inventory drawn up in 1854 and expanded the collection with acquisitions and contemporary works from her National Exhibitions of Fine Arts that she began in 1856, making it the first national Spanish public art museum with state-owned works.
In 1862, the art critic and historian Gregorio Cruzada Villaamil became deputy director of the museum, and he was the first to properly review and catalog the inventories of the collection.
Of the 1733 paintings that appear in the 1854 inventory, he cataloged only 603, noting that works previously in the collection of Infante Sebastian Gabriel seized in 1835 had been returned to him in 1861.
By decrees of the regency government of November 25, 1870 and March 22, 1872, the Museo de la Trinidad was closed down and its collections were transferred to the Prado (the former 'Royal Museum').
Among the most valuable pieces from the Museo de la Trinidad were those by El Greco, Pedro Berruguete, Juan Bautista Maíno, and Flemish paintings.