Ag processing by intracellular proteases and peptidases and epitope presentation are critical for recognition of pathogen-infected cells by CD8+ T lymphocytes. First-generation HIV protease inhibitors (PIs) alter proteasome activity, but the effect of first- or second-generation PIs on other cellular peptidases, the underlying mechanism, and impact on Ag processing and epitope presentation to CTL are still unknown. In this article, we demonstrate that several HIV PIs altered not only proteasome but also aminopeptidase activities in PBMCs. Using an in vitro degradation assay involving PBMC cytosolic extracts, we showed that PIs altered the degradation patterns of oligopeptides and peptide production in a sequence-specific manner, enhancing the cleavage of certain residues and reducing others. PIs affected the sensitivity of peptides to intracellular degradation, and altered the kinetics and amount of HIV epitopes produced intracellularly. Accordingly, the endogenous degradation of incoming virions in the presence of PIs led to variations in CTL-mediated killing of HIV-infected cells. By altering host protease activities and the degradation patterns of proteins in a sequence-specific manner, HIV PIs may diversify peptides available for MHC class I presentation to CTL, alter the patterns of CTL responses, and provide a complementary approach to current therapies for the CTL-mediated clearance of abnormal cells in infection, cancer, or other immune disease.