Previous experiments using enzyme inhibitors, cell lysates, and purified enzyme have suggested that puromycin-sensitive aminopeptidase (PSA) plays a role in creating and destroying MHC class I-presented peptides although its precise contribution to these processes is unknown. To examine the importance of this enzyme in MHC class I Ag presentation, we have generated PSA-deficient mice and cell lines from these animals. PSA-deficient mice are smaller and do not reproduce as well as wild type mice. In addition, dendritic cells from PSA-deficient mice display more MHC class I molecules on the cell surface, suggesting that PSA normally limits Ag presentation by destroying certain peptides in these key APCs. Surprisingly, MHC class I levels are not altered on other PSA-deficient cells and the processing and presentation of peptide precursors in PSA-deficient fibroblasts is normal. Moreover, PSA-deficient mice have normal numbers of T cells in the periphery, and respond as well as wild type mice to eight epitopes from three viruses. These data indicate that PSA may play a role in limiting MHC class I Ag presentation in dendritic cells in vivo but that it is not essential for generating most MHC class I-presented peptides or for stimulating CTL responses to several Ags.