Murine macrophages can be activated to produce nitric oxide (NO) and superoxide and these two radicals can react to form peroxynitrite, a powerful oxidant which may be involved in parasite killing. We now show that murine macrophages activated with zymosan and interferon-gamma (ZYM/IFN-gamma) produced both superoxide (peaking 1-2 h after stimulation, then rapidly declining) and NO (barely detectable at 6 h, peaking by 24 h). Macrophages activated with ZYM alone produced only superoxide, while stimulation with lipopolysaccharide (LPS) and IFN-gamma induced NO but not superoxide. Cells stimulated with ZYM/IFN-gamma or LPS/IFN-gamma killed Leishmania major to a similar degree, an effect that was completely blocked by the addition of N-iminoethyl-L-ornithine. However, macrophages stimulated with ZYM alone were unable to kill L. major. S-nitroso-acetyl-penicillamine, which releases NO, was highly leishmanicidal when added directly to the parasites. 3-morpholino-sydnonimine hydrochloride which releases both NO and superoxide simultaneously, was also efficient at killing L. major and this cytotoxicity was greatly enhanced by the addition of superoxide dismutase. Finally, authentic peroxynitrite failed to induce any cytotoxic effect, even at a high concentration. Thus macrophages can produce either NO, superoxide or both, depending on the stimulus. However, the killing of L. major is dependent only on the production of NO.