We have compared by SDS-PAGE Western blotting the molecules detected by two human monoclonal antibodies, C-OU1 and 16.88. The antibodies have previously been shown to detect a cytoplasmatic antigen with an Mr of 43 kD present in colon adenocarcinoma cell lines and in colon cancer tissues. We now demonstrate that these antibodies differ significantly in their fine specificity, resulting in a quite dissimilar tumor selectivity. The antibody 16.88, in addition to reactivity with the 43-kD molecule, also recognizes a 190-kD molecule present both in melanoma cells and in cells previously reported as 16.88 antigen positive. The 16.88 antibody does not detect a 43-kD molecule in extracts of melanoma cells. The 190-kD component was not detectable in hepatoma or mamma carcinoma cells, both of which showed presence of the 43-kD molecule. The C-OU1 antibody shows no reactivity with the 190-kD molecule in any of the cells tested or with other proteins in melanoma cells. Radiolabeled 16.88 antibody shows better localization to melanoma cancer than to colon cancer xenograft transplanted onto nude mice. These findings indicate the presence of a tumor-associated antigen not previously described and have obvious implications for potential clinical uses of the antibodies.