Two HLA-B*3501 binding self-peptldes, LPFDFTPQY (37F) and LPGPKFLQY (28H), were Isolated from HLA-B*3501 molecules expressed by cultured human B lymphold cells. Both sequences were consistent with previously reported motifs of HLA-B*3501 binding peptides which carry prollne at position 2 and tyroslne at position 9 as anchor residues. Direct binding of these peptides to HLA-B*3501 molecules was quantitated by flow cytometry analysis of RMA-S cells transfected with the HLA-B*3501 gene (RMA-S-B*3501). Both 37F and 28H peptides bound effectively to HLA-B*3501 molecules. Substitution of amlno acids at position 2 and/or 9 of HLA-B*3501 binding peptides markedly reduced their binding to HLA-B*3501 molecules. These results indicate that two anchor residues, prollne at position 2 and tyroslne at position 9 are critical in binding of peptides to HLA-B*3501 molecules. Insertion of up to four glycine residues at position 8 of the peptide 37F did not affect Its binding affinity to HLA-B*3501 molecules. These results indicate that long peptides can effectively bind to HLA class I molecules provided that anchor residues are conserved.