Conditions were established to induce rapid clonal growth of melanocytes from newborn foreskin. Surface antigen expression was analyzed using monoclonal antibodies derived by immunization of mice with melanoma cell, melanocyte, and placental membrane preparations. Unlike resting melanocytes in normal skin, cultured melanocytes expressed most major melanoma-associated antigens tested, e.g., nerve growth factor receptor, proteoglycan, transferrin-related Mr 97,000 protein antigen, Mr 120,000 protein, and gangliosides 9-O-acetyl GD3 and GD3. HLA-DR antigen and ganglioside GD2 were expressed at very low levels or not expressed. After several subpassages, most melanocyte cultures, including clones and melanocytes, initially sorted by rosetting with monoclonal antibody to nerve growth factor receptor, lost their characteristic bipolar morphology and expression of nerve growth factor receptor and Mr 97,000 antigen but continued to express high molecular weight proteins such as proteoglycan, Mr 130,000/105,000 and 120,000 antigen. The few melanocyte cultures that did maintain their characteristic bipolar to spindle morphology continued to express all melanoma-associated antigens and even began to express HLA-DR antigens. Melanocytes cultured in the presence of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate also maintained their bipolar morphology, were often pigmented, and continued to express melanoma-associated antigens for several passages; they did not express HLA-DR antigen. Our studies indicate that rapidly proliferating melanocytes in culture undergo antigenic changes associated with malignancy.