Intracellular proteins are degraded in the antigen processing pathway to generate peptide-loaded MHC I complexes (pMHC I) for immune surveillance. The characteristics of the final pMHC I are clear but those of their precursors and their potential binding partners remain poorly defined. By using a unique method to biochemically detect preprocessed ovalbumin-derived antigenic peptides, we find that cells generate large, C-terminally extended proteolytic intermediates that are associated with the α isotype of hsp90 chaperone. Knockdown of hsp90α expression by siRNA resulted in the loss of these intermediates and decreased presentation of the final pMHC I on the cell surface. Generation of pMHC I was also inhibited by knockdown of the cochaperone CHIP that interacts with heat shock proteins, ubiquitinates their clients, and delivers them to the proteasome. Thus, hsp90α can serve as a chaperone for precursors of pMHC I at an early stage in the antigen processing pathway.