The capacity of 15 separate populations of mouse peritoneal macrophages to generate and release H2O2 (an index of oxidative metabolism) was compared with their ability to inhibit the intracellular replication of virulent Toxoplasma gondii. Resident macrophages and those elicited by inflammatory agents readily supported toxoplasma multiplication and released 4-20X less H2O2 than macrophages activated in vivo by systemic infection with Bacille Calmette-Guérin or T. gondii, or by immunization with Corynebacterium parvum. Immunologically activated cells consistently displayed both enhanced H2O2 production and antitoxoplasma activity. Exposure to lymphokines generated from cultures of spleen cells from T. gondii immune mice and toxoplasma antigen preserved both the antitoxoplasma activity and the heightened H2O2 release of toxoplasma immune and immune-boosted macrophages, which otherwise were lost after 48-72 h of cultivation. In vitro activation of resident and chemically-elicited cells by 72 h of exposure to mitogen- and antigen-prepared lymphokines, conditions that induce trypanocidal (5) and leishmanicidal activity (14), stimulated O2- and H2O2 release, and enhanced nitroblue tetrazolium reduction in response to toxoplasma ingestion. Such treatment, however, failed to confer any antitoxoplasma activity, indicating that intracellular pathogens may vary in their susceptibility to macrophage microbicidal mechanisms, including specific oxygen intermediates. In contrast, cocultivating normal macrophages with lymphokine plus heart infusion broth for 18H rendered these cells toxoplasmastatic. This in vitro-acquired activity was inhibited by scavengers of O2-, H2O2, OH., and 1O2, demonstrating a role for oxidative metabolites in lymphokine-induced enhancement of macrophage antimicrobial activity. These findings indicate that augmented oxidative metabolism is an consistent marker of macrophage activation, and that oxygen intermediates participate in the resistance of both in vivo- and vitro-activated macrophages toward the intracellular parasite, T. gondii.