T-cell recognition of peptides that are bound and presented by class I major histocompatibility complex molecules is highly specific. At present it is unclear what role class I peptide binding plays relative to T-cell receptor specificity in determination of immune recognition. A previous study from our group demonstrated that the HLA-A2.1 molecule could bind to 25% of the members of a panel of unrelated synthetic peptides as assessed by a functional peptide competition assay. To determine the peptide-binding specificity of another HLA class I molecule, we have 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-A2.1 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.