Sera of 106 normal adult men were tested for antibodies reacting with cell surface antigens of three established lines of cultured malignant melanoma. Positive reactions with a protein A assay for IgG antibodies were extremely rare (1-2%). The frequency of positive reactions with assays for IgM antibodies was higher: 5-15% in immune adherence assays and 55-82% in anti-C3 mixed hemadsorption assays. After low-titered sera and sera reacting with fetal calf serum components, conventional alloantigens, and widely distributed class 3 antigens were excluded, sera from seven individuals (one with IgG antibody and six with IgM antibodies) were selected for detailed analysis. The serum containing the IgG antibody came from a healthy 65-year-old Caucasian man; titers of antibody in his serum ranged from < 1/10 to 1/40,000 in tests with different melanoma cell lines. This IgG antibody identifies a differentiation antigen of melanocytes, provisionally designated Mel 1, that distinguishes two classes of melanomas: 22 melanoma cell lines typed Mel 1+ and 17 types Mel 1-. Mel 1 is expressed by fetal fibroblasts but not adult fibroblasts and can be found on a proportion of cultured epithelial cancer cell lines (5 out of 23) but not on glioma or B-cell lines. The melanoma antigens detected by the naturally occurring IgM antibodies are serologically unrelated to Mel 1 but, like Mel 1, appear to be differentiation antigens that distinguish subsets of melanoma. These IgM antibodies detect antigens that are identical or closely related to the AH antigen, a melanoma surface antigen that was initially defined by autologous antibody in a patient with melanoma. In view of the immunogenicity of both Mel 1 and the AH antigens in humans and their occurrence on more than 50% of melanomas, it remains to be seen whether antibody to these antigens can be elicited by specific vaccination of seronegative melanoma patients and whether this will have an influence on the clinical course of the disease.