To detect viral infections and tumors, CD8 T lymphocytes monitor cells for the presence of antigenic peptides bound to MHC class I molecules. The majority of MHC class I-presented peptides are generated from the cleavage of cellular and viral proteins by the ubiquitin-proteasome pathway. Many of the oligopeptides produced by this process are too long to stably bind to MHC class I molecules and require further trimming for presentation. Cytosolic aminopeptidases such as leucine aminopeptidase (LAP), which is IFN-inducible, Bleomycin Hydrolase (BH), and puromycin-sensitive aminopeptidase (PSA) can trim precursor peptides to mature epitopes and have been thought to play an important role in antigen presentation. To examine the role of these aminopeptidases in generating MHC class I peptides in vivo, we generated mice deficient in LAP or PSA, as well as cell lines deficient in LAP, PSA, or BH. LAP mutant mice and cells are viable and grow normally, whereas PSA mutant mice are smaller than their wild-type and heterozygote littermates, are subfertile as adults, and are subviable as embryos. The trimming of peptides in LAP-deficient cells is not reduced under basal conditions or after stimulation with IFN. Similarly, there is no reduction in presentation of peptides from precursor or full length antigen constructs or in the overall supply of peptides from cellular proteins to MHC class I molecules, even after stimulation with IFN. After viral infection, LAP-deficient mice generate normal CTL responses to seven epitopes from three different viruses. Similarly, PSA deficient mice and BH deficient mice generate normal CTL responses to viral epitopes. These data demonstrate that LAP, BH, and PSA are not essential enzymes for generating most MHC class I-presented peptides and reveal redundancy in the function of cellular aminopeptidases in most cell types.