Pigmented melanoma cells and cultured melanocytes express a differentiation-related glycoprotein designated as pigmentation-associated antigen (PAA) of Mr 70,000-80,000. As described previously, PAA was initially defined by reactivity with antibodies in the serum of a patient with melanoma. Here we describe the production and characterization of a mouse monoclonal antibody to PAA. This antibody (TA99, an IgG2a) was shown by sequential immunoprecipitation experiments to react with the same component as the human antibody. Ab TA99 immunoprecipitated PAA from lysates of cells radiolabeled with [35S]methionine, [3H]glucosamine, [3H]fucose, and [3H]mannose as well as 125I. Using Ab TA99, the distribution of PAA was examined in frozen sections of 19 normal tissues and quantitatively in 68 tissue culture cell lines. In frozen sections, only melanin-containing cells were positive, including epithelial cells in the basal layer of the epidermis, in which pigment originates from melanocytes by transfer of melanosomes, and pigmented cells of the eye. In tissue culture cell lines, only pigmented melanoma cells were positive. PAA is an intracellular antigen, with a distribution very similar to that of melanosomes. This evidence confirms the close association of PAA with melanin production, and suggests that PAA may be a melanosome component. PAA was shown to be different from tyrosinase, the enzyme involved in melanin synthesis, but it was found to be identical to the previously recognized glycoprotein, gp75, characteristic of pigmented melanomas and melanocytes.