L-Arginine-derived nitric oxide (NO) acts as an inter- and intra-cellular signal molecule in many mammalian tissues including brain, where it is formed by a flavin-containing Ca2+/calmodulin-requiring NO synthase with NADPH, tetrahydrobiopterin (H4biopterin) and molecular oxygen as cofactors. We found that purified brain NO synthase acted as a Ca2+/calmodulin-dependent NADPH:oxygen oxidoreductase, catalysing the formation of hydrogen peroxide at suboptimal concentrations of L-arginine or H4biopterin, which inhibited the hydrogen peroxide formation with half-maximal effects at 11 microM and 0.3 microM respectively. Half-maximal rates of L-citrulline formation were observed at closely similar concentrations of these compounds, indicating that the NO synthase-catalysed oxygen activation was coupled to the synthesis of L-citrulline and NO in the presence of L-arginine and H4biopterin. N omega-Nitro-L-arginine, its methyl ester and N omega-monomethyl-L-arginine inhibited the synthesis of L-citrulline from L-arginine (100 microM) with half-maximal effects at 0.74 microM, 2.8 microM and 15 microM respectively. The N omega-nitro compounds also blocked the substrate-independent generation of hydrogen peroxide, whereas N omega-monomethyl-L-arginine did not affect this reaction. According to these results, activation of brain NO synthase by Ca2+ at subphysiological levels of intracellular L-arginine or H4biopterin may result in the formation of reactive oxygen species instead of NO, and N omega-nitro-substituted L-arginine analogues represent useful tools to effectively block NO synthase-catalysed oxygen activation.