Three mouse monoclonal antibodies (mAbs) recognizing antigens of human melanocytic cells were used to study a large panel of cultured normal and tumor cells and fresh tissues. Two of the monoclonal antibodies (designated TA99 and CF21) detect melanosomal antigens, whereas mAbC350 recognizes a cell surface antigen. Among cultured cells the three mAbs reacted exclusively with normal melanocytes and pigmented melanomas, but not with nonpigmented melanomas or cells of other lineage. Immunohistochemical assays using mAbTA99 and mAbCF21 on fresh-frozen sections from a large panel of normal tissues revealed a characteristic pattern of reactivity restricted to pigmented cells. mAbC350 did not react with any normal tissues. All nevi and primary melanoma specimens and 93% of metastatic melanomas were reactive with at least one of these three monoclonal antibodies. No reactivity was found with 62 nonmelanoma tumors. An inverse correlation was observed between TA99 expression and stage of tumor progression. These markers are useful for studies of melanocyte differentiation and malignant transformation, subsetting of melanocytic lesions, and identification of tumors of melanocytic origin.