Superoxide (O_2⨪) and nitric oxide (NO) act to kill invading microbes in phagocytes. In macrophages NO is synthesized by inducible nitric oxide synthase (iNOS, NOS 2) from l-arginine (l-Arg) and oxygen; however, O_2⨪ was thought to be produced mainly by NADPH oxidase. Electron paramagnetic resonance (EPR) spin trapping experiments performed in murine macrophages demonstrate a novel pathway of O_2⨪ generation. It was observed that depletion of cytosolic l-Arg triggers O_2⨪ generation from iNOS. This iNOS-mediated O_2⨪ generation was blocked by the NOS inhibitor N-nitro-l-arginine methyl ester or by l-Arg, but not by the noninhibitory enantiomer N-nitro-d-arginine methyl ester. In l-Arg-depleted macrophages iNOS generates both O_2⨪ and NO that interact to form the potent oxidant peroxynitrite (ONOO⁻), which was detected by luminol luminescence and whose formation was blocked by superoxide dismutase, urate, or l-Arg. This iNOS-derived ONOO⁻ resulted in nitrotyrosine formation, and this was inhibited by iNOS blockade. iNOS-mediated O_2⨪ and ONOO⁻ increased the antibacterial activity of macrophages. Thus, with reduced l-Arg availability iNOS produces O_2⨪ and ONOO⁻ that modulate macrophage function. Due to the existence of l-Arg depletion in inflammation, iNOS-mediated O_2⨪ and ONOO⁻ may occur and contribute to cytostatic/cytotoxic actions of macrophages.