The ability of HLA-A2 and HLA-B37 molecules to bind a set of randomly selected peptides was studied in order to learn more about the specificity of the peptide binding site in class I molecules. It was shown that: 1) the HLA-A2.1 molecule can bind a broad spectrum of peptides; and 2) T cells selected for the ability to recognize one peptide plus a class I molecule can actually recognize an unrelated peptide presented by the same class I molecule. To determine if another class I molecule would bind the same or a different set of peptides, we studied the binding specificity of HLA-B37 with the same set of peptides. We examined the capacity of this panel of peptides to compete for the presentation of influenza virus nucleoprotein peptide NP-(335-350) by HLA-B37 to NP-peptide-specific HLA-B37-restricted cytotoxic T-lymphocyte lines. Forty-two percent of peptides tested were capable of inhibiting NP-(335-350) presentation by HLA-B37. Remarkably, none of these HLA-B37 binding peptides belong to the subset that was previously shown to bind to the HLA-A2.1 molecule. Only the Np (335-350) peptide was capable of binding to both HLA-A2.1 and HLA-B37. These findings demonstrate that the peptide-binding specificities of HLA-B37 and HLA-A.1 are largely nonoverlapping and suggest that, from the universe of peptides, individual HLA class I molecules can bind to clearly distinct subsets of these peptides. Amino acid substitutions were made at key sites in the beta-pleated floor of the HLA-A2 peptide binding groove in order to study their effect on recognition by allospecific and peptide specific CTL. The results obtained demonstrated that amino acid side chains in the floor of the peptide binding groove can have a major functional role in determining the formation and conformation of the peptide-A2 complex that is recognized by peptide-specific CTL.