The activity of a complement-dependent cytotoxic antibody in the sera of 21 melanoma patients was investigated using a microcytotoxicity assay. Heat-inactivated sera were caused to react against mechanically dispersed fresh tumor cells in the presence of exogenous blood group AB complement. Cytotoxicity was evaluated relative to pooled normal sera as a control. Sera were cytotoxic against autochthonous tumor cells in 9 of 10 patients with localized or regional melanoma and in 1 of 11 patients with disseminated metastases. Cytotoxicity of sera was unrelated to size of tumor burden. Six of 7 antibody-positive sera (autochthonous system) were noncytotoxic to between 2 and 7 different allogeneic melanoma tumor cell preparations. Immunological reactivity of the cytotoxic antibody-positive and -negative groups was similar with respect to their capacity to be sensitized to dinitrochlorobenzene, produce positive skin tests to microbial antigens, and produce antibodies to typhoid vaccination; serum immunoglobulins were comparable. These results support the reported findings of the presence of cytotoxic antibody in the sera of melanoma patients without disseminated metastases.