Sera from melanoma patients undergoing immunization with three distinct immunogens were evaluated for development of IgG antibody specific for the melanoma tumor-associated antigens (TAAs) GD3 and 250-kd glycoprotein (high molecular weight; HMW). Of 99 patients receiving either irradiated allogeneic melanoma cells admixed with bacillus Calmette-Guérin, vaccinia viral oncolysate melanoma cell membranes, or cholesterol hemisuccinate-modified melanoma cells, 12 were determined to have responded against GD3 and 17 against the HMW TAA. This reactivity was measured using both direct binding to purified TAA and specific inhibition of binding of murine monoclonal antibodies R24 and Me-1-14 for GD3 and HMW TAA, respectively. Preimmune sera from these patients did not react with these TAAs, nor did preimmune sera or follow-up sera from melanoma patients electing not to receive immunotherapy. These results suggest that melanoma patients can be immunized against these TAAs as presented on the melanoma cell membrane in an immunotherapy setting and that immunization using purified TAAs might likewise be feasible.