The immunogenicity of gangliosides found on human melanoma cells was determined from sera of 26 melanoma patients who were immunized every 1-4 weeks for 4 months with tumor-cell vaccine (TCV) prepared from cultured melanoma cells. Total lipid-bound sialic acid in the gangliosides isolated from TCV was 0.38 mumol/10(8) cells, and was distributed as follows: 44.8% to GM3, 44.2% to GD3, 5.6% to GM2, and 4.6% to GD2. Sera were tested at monthly intervals for antibodies to each ganglioside by ELISA with purified gangliosides as the antigen source. The immunologic specificity of the antibody was confirmed by absorption tests. None of the 26 patients had detectable anti-GM3, anti-GD3, or anti-GD2 antibodies before immunization, although anti-GM2 antibody was detected in 3 patients. After immunization, 2 patients developed IgM anti-GD2, 10 developed IgM anti-GM2, and 2 developed IgG anti-GM2 antibodies. No patient developed detectable anti-GM3 or anti-GD3 antibodies. These results indicate that both GD2 and GM2 expressed on human melanoma cells are immunogenic in humans, although GM2 appears to be more immunogenic. The other two gangliosides, GM3 and GD3, are present in human sera and in human normal tissues, and thus immunologic tolerance may have been established against these gangliosides. Alternatively, circulating GM3 and GD3 may have neutralized anti-GM3 and anti-GD3 antibodies, if any were induced by TCV immunization.