The function of MHC class I molecules is to bind and present antigenic peptides to cytotoxic T cells. Here, we report that class I-restricted peptide presentation is strongly pH dependent. The presentation of some peptides was enhanced at acidic pH, whereas the presentation of others was inhibited. Biochemical peptide-MHC class I binding assays demonstrated that peptide-MHC class I complexes are more stable at neutral pH than at acidic pH. We suggest that acid-dependent peptide dissociation can generate empty class I molecules and that the resulting binding potential can be exploited by a subset of peptide-MHC class I combinations, in some cases leading to considerable peptide exchange. We further speculate that the relative instability of peptide-class I complexes under acidic conditions may affect the outcome of class I-restricted Ag presentation, as less stably associated peptides may dissociate from class I during passage of the acidic trans-Golgi network, and therefore may not be presented. Finally, our results may in part explain how endocytosed proteins can be presented by MHC class I molecules to cytotoxic T cells.