MART-1 is expressed ir both normal and neoplastic cells of melanocytic origin. Peripheral blood mononuclear cells (PBMC) from melanoma patients recognize and lyse tumor cells after repetitive in vitro stimulation with the immunodominant peptide MART-1(27-35). In this study, we compared the characteristics of the cytotoxic T lymphocyte (CTL) response to MART-1 in PBMC from 13 HLA-A2 melanoma patients with PBMC from 9 normal healthy donors stimulated in vitro with MART-1(27-35) (AAGIGILTV) or FluM1(58-66) (GILGFVFTL) peptides. The expansion rate among CTLs from different patients was variable and did not correlate with the development of specificity against the MART-1(27-35) or FluM1(58-66) peptides. Specific anti-MART-1(27-35) cytotoxicity could be generated in 13 of 13 melanoma patients but only in 5 of 9 healthy donors (p < 0.001). Anti-FluM1(58-66) activity could be generated in six of seven melanoma patients and six of seven healthy donors. Specific activity against MART-1(27-35), but not FluM1(58-66), was detectable significantly earlier after repetitive in vitro stimulation in melanoma patients (22.7 +/- 2.0 days compared with 32.7 +/- 1.7 days for healthy donors, p < 0.01). This report provides the first evidence of an enhanced level of sensitization of tumor-bearing hosts compared with normal individuals against a differentiation antigen shared by tumor and normal cells of the same lineage. These findings may have important implications for delineating events involved in the biology of tumor rejection naturally or in response to active specific immunotherapy.