Peptides derived from melanocyte differentiation antigens have been identified as targets for MHC class I‐restricted cytolytic T lymphocytes (CTLs) in human melanoma. Regression of antigen‐expressing tumors as well as selection of antigen‐loss variants in the presence of antigen‐specific CTLs have previously been reported. In the present study, we determined the expression of the melanocyte differentiation antigens Melan A/MART‐I and tyrosinase by mRNA analysis and by immunohistochemical staining with the monoclonal antibodies (MAbs) A103 and T311. Co‐expression of Melan A/MART‐I and tyrosinase was detected by both methods in 18/20 melanomas tested. However, immunohistochemistry provided additional information on intensity and microheterogeneity of antigen expression that cannot be detected by mRNA analysis as a molecular basis for the escape from CTL recognition of antigen‐negative tumor cells. Comparative analysis of repeated biopsies of metastatic lesions in 5 HLA‐A2⁺ patients showed a gradual loss of Melan A/MART‐I expression in 4/5 and of tyrosinase in 2/5 samples in association with tumor progression. However, 3 of these patients had growing antigen‐positive tumors in the presence of antigen‐specific CTLs. This led us to assess the expression of MHC class I, the essential restriction element for CTL recognition, and of HLA‐A2. We found an unexpectedly high frequency of MHC class I‐negative tumors (9/20). Loss of MHC class I expression was detected in 3/5 progressive tumors and isolated loss of HLA‐A2 in 1/5 tumors. Our results suggest that strategies enhancing the expression of MHC class I and tumor‐associated antigens need to be considered in attempts at making vaccination more effective. Int. J. Cancer, 71:142–147, 1997. © 1997 Wiley‐Liss, Inc.