Human neutophils stimulated with phorbol myristate acetate were able to rapidly destroy autologous red blood cell targets. Neutrophil-mediated cytotoxicity was related to phorbol myristate acetate concentration and neutrophil number. The ability of stimulated neutrophils to lyse red blood cell targets was markedly impaired by catalase or superoxide dismutase but not by heat-inactivated enzymes or albumin. Despite a simultaneous requirement for O2.- and H2O2 in the cytotoxic event, a variety of OH. and 1O2 did not effect cytolysis. The myeloperoxidase inhibitor cyanide did not reduce red blood destruction, while azide consistently impaired cytolysis. The inability of cyanide to reduce cytotoxicity coupled with the protective effect of superoxide dismutase suggests that cytotoxicity is independent of the classic myeloperoxidase-H2O2-halide system. We propose that neutrophils, stimulated with phorbol myristate acetate, generate O2.- and H2O2, which play an integral role in a novel cytotoxic mechanism.