Melanoma‐reactive human cytotoxic T lymphocytes (CTLs) mediate tumor regression in vivo through specific recognition of MHC‐associated peptide epitopes, many of which are encoded by the melanocytic tissue differentiation proteins gp100/Pmel17 and MART‐1/Melan‐A. Vaccines using these peptides may induce protective or therapeutic immunity against melanoma. Rational design of such approaches is aided by a clear understanding of the identity of these antigenic peptides; however, most CTL epitopes described to date were identified indirectly. Especially where these peptides may be used in human clinical trials for the treatment or prevention of cancer, there is substantial need for direct evaluation of HLA‐A*0201‐associated peptides from MART‐1 and gp100 that are naturally processed and presented. To that end, we have isolated peptides directly from HLA‐A*0201 molecules of human melanoma cells and have determined that naturally processed epitopes for HLA‐A*0201‐restricted, melanoma‐reactive CTLs include the nonamers MART‐1_27–35 (AAGIGILTV), gp100_154–162 (KTWGQYWQV), gp100_209–217 (ITDQVPFSV) and gp100_280–288 (YLEPGPVTA) and the decamer gp100_476–485 (VLYRYGSFSV). Among these, the one that appears to be most abundant at the cell surface is gp100_154–162 (KTWGQYWQV). The others are among the less abundant peptides. HLA‐A*0201‐restricted CTLs from one melanoma patient who has survived metastatic disease recognized MART‐1_27–35 (AAGIGILTV), gp100_280–288 (YLEPGPVTA) and gp100_154–162 (KTWGQYWQV) and were cross‐reactive on longer peptides that contained these nonamer sequences. These peptides, identified by both an indirect genetic approach and by a direct peptide approach, can be used for tumor vaccine strategies with confidence that they are identical to the naturally processed peptide epitopes presented at the surface of melanoma cells in association with HLA‐A*0201 molecules. Int. J. Cancer 82:669–677, 1999. © 1999 Wiley‐Liss, Inc.