Neuronal nitric-oxide synthase (NOS I) in the absence of L-arginine has previously been shown to generate superoxide (O-2) (Pou, S., Pou, W. S., Bredt, D. S., Snyder, S. H., and Rosen, G. M. (1992) J. Biol. Chem. 267, 24173-24176). In the presence of L-arginine, NOS I produces nitric oxide (NO.). Yet the competition between O2 and L-arginine for electrons, and by implication formation of O-2, has until recently remained undefined. Herein, we investigated this relationship, observing O-2 generation even at saturating levels of L-arginine. Of interest was the finding that the frequently used NOS inhibitor NG-monomethyl L-arginine enhanced O-2 production in the presence of L-arginine because this antagonist attenuated NO. formation. Whereas diphenyliodonium chloride inhibited O-2, blockers of heme such as NaCN, 1-phenylimidazole, and imidazole likewise prevented the formation of O-2 at concentrations that inhibited NO. formation from L-arginine. Taken together these data demonstrate that NOS I generates O-2 and the formation of this free radical occurs at the heme domain.