Neuronal nitric oxide synthase (nNOS) is a modular enzyme which consists of a flavin-containing reductase domain and a heme-containing oxygenase domain, linked by a stretch of amino acids which contains a calmodulin (CaM) binding site. CaM binding to nNOS facilitates the transfer of NADPH-derived electrons from the reductase domain to the oxygenase domain, resulting in the conversion of l-arginine to l-citrulline with the concomitant formation of a guanylate cyclase activating factor, putatively nitric oxide. Numerous studies have established that peroxynitrite-derived nitrogen oxides are present following nNOS turnover. Since peroxynitrite is formed by the diffusion-limited reaction between the two radical species, nitric oxide and O₂^•-, we employed the adrenochrome assay to examine whether nNOS was capable of producing O₂^•- during catalytic turnover in the presence of l-arginine. To differentiate between the role played by the reductase domain and that of the oxygenase domain in O₂^•- production, we compared its production by nNOS against that of a nNOS mutant (CYS-331), which was unable to transfer NADPH-derived electrons efficiently to the heme iron under special conditions, and against that of a flavoprotein module construct of nNOS. We report that O₂^•- production by nNOS and the CYS-331 mutant is CaM-dependent and that O₂^•- production can be modulated by substrates and inhibitors of nNOS. O₂^•- was also produced by the reductase domain of nNOS; however, it did not display the same CaM dependency. We conclude that both the reductase and oxygenase domains of nNOS produce O₂^•-, but that the reductase domain is both necessary and sufficient for O₂^•- production.