Guinea pig peritoneal macrophages (GPPM) exhibited enhanced production of O2- and H2O2, and cytolytic activity toward erythrocytes, in response to reagents such as 12-O-tetradecanoyl-phorbol-13-acetate (TPA), its methylated derivative 4-O-MeTPA, Con A, wheat germ agglutinin (WGA), and opsonized zymosan. In order to examine the possible role of oxidative burst products such as O2- and H2O2 in the cytolytic process, we used reagents and enzymes which influence the balance of O2- and H2O2 outside and inside the GPPM cells. Macrophage-mediated cytolysis (MMC) of erythrocytes in the presence of the activators and modulators was assessed by 51Cr release assay. MMC activated by TPA and 4-O-MeTPA was inhibited by scavengers of H2O2 such as catalase and α-tocopherol, and was augmented by the catalase inhibitor 3-amino-1,2,4-triazole, and by horseradish peroxidase. TPA- and 4-O-MeTPA-activated MMC was only partially inhibited by the O2- scavenger cytochrome c and the enzyme superoxide dismutase and unaffected by cytochalasin D (an inhibitor of phagocytosis). MMC activated by the lectins Con A and WGA was unaffected by the scavengers and enzymes used, but markedly inhibited by cytochalasin D. Activation of MMC by TPA, WGA, and phagocytosis of opsonized zymosan, as well as O2- and H2O2 generation triggered by these reagents, were markedly inhibited by chlorpromazine. The results indicate that GPPM-mediated cytolysis activated by lectins, phorbol ester derivatives, and phagocytosis of opsonized zymosan, is dependent on the generation of oxidative burst products, mainly H2O2. TPA- or 4-O-MeTPA-activated MMC is mainly an extracellular event, while lectin-activated MMC may take place within the macrophages.