A panel of autologous cytolytic T lymphocyte (CTL) clones have been isolated from blood lymphocytes of a melanoma patient after in vitro stimulation with autologous tumor cells. We previously reported the molecular definition of three distinct antigens recognized by some of these CTL clones. We describe here, the identification of a fourth antigenic peptide expressed by this melanoma line and recognized by a CTL clone restricted by HLA-B*3503. The antigenic peptide, which is nine-amino acid long, has the sequence LPHSSSHWL and is derived from melanocyte differentiation antigen gp100. As HLA-B35 is one of the most frequent HLA-B alleles, being present in 20% of the Caucasian individuals, this peptide may be a good target for peptide-based immunotherapy of melanoma.