Antigenic peptides derived from differentiation antigens of the melanocyte lineage were recently identified in human melanomas as targets for MHC-restricted cytotoxic T lymphocytes (CTL). CTL directed against peptides derived from the Melan A/MART-1, tyrosinase and gp100/Pmel17 antigens can be detected in melanoma patients and in healthy controls. The presence of defined antigenic peptides and corresponding precursor CTL in patients with metastatic melanoma opens perspectives for the development of antigen-specific tumor vaccines. In this study, we examined the expression of Melan A/MART-1, tyrosinase and gp100lPmel17 in fresh melanoma tissues of HLA-A2+ patients and the spontaneous CTL reactivity against antigenic peptides derived from these antigens. Our results demonstrate an inverse correlation of antigen expression and CTL response to Melan A/MART-1 and tyrosinase in patients with metastatic melanoma. In 2 patients with advanced disease, CTL responses against Melan A/MART-1 and tyrosinase were induced by intradermal immunization with synthetic nona- or deca-peptides derived from these antigens. Metastases increasing in size over time showed a loss of Melan A/MART-1 expression in the presence of CTL in one patient. The regression of a metastasis with persistent tyrosinase expression was observed in the other patient after the induction of CTL, reactive against tyrosinase. We conclude that CTL responses against melanocyte differentiation antigens may mediate regression of antigen-positive tumors and select for antigen-loss variants in vivo.