The hybridoma 653.40S, constructed with splenocytes from an inbred BALB/c mouse immunized with cultured human melanoma cells, secreted an antibody that had been shown to recognize an antigenic determinant restricted to human melanoma cell lines. The monoclonal antibody (MoAb) 653.40S showed immunoprecipitation of two glycopolypeptides synthesized by melanoma cells, one with the apparent molecular weight of 280,000 and the other one with a molecular weight larger than 500.000. These two glycopolypeptides were not bridged by disulfide bonds and were peripheral rather than integral to the plasma cell membrane. Comparison of the reactivity of cells of the melanocyte lineage with the MoAb 653.40S and with the MoAb Q5/13 to human Ia-like antigens showed that the former reacted with proliferating melanocytes and melanoma cells, whereas the latter reacted only with melanoma cells. The MoAb 653.40S did not react with a large variety of surgically removed normal and tumor tissues except for some instances of basal cell and squamous cell carcinomas. These results suggested that double staining of pigmented skin lesions with the MoAb 653.40S and with an MoAb to Ia-like antigens may help to solve controversial diagnosis of melanoma. Furthermore, the MoAb 653.40S may be useful for radioimaging and immunotherapy of melanoma.