Nitric oxide (NO) produced by the endothelial isoform of nitric oxide synthase (NOS III) is a key determinant of the anti-atherosclerotic properties of the endothelium. Recent in vivo studies suggest that NOS III may also be a source of superoxide production, which would limit its role as a NO-producing enzyme. In the current study we examined both the NO and the superoxide generating potential of recombinant NOS III obtained from a baculovirus/Sf9 expression system. Using lucigenin chemiluminesence we could indeed demonstrate (superoxide dismutase inhibitable) superoxide production by NOS III. This superoxide production was not affected by administration of L-arginine, but could be inhibited dose-dependently by the co-factor tetrahydrobiopterin (BH4). BH4 also dose dependently decreased superoxide generation by hypoxanthine/xantine oxidase suggesting a direct antioxidant effect. Superoxide generation by NOS III could be completely inhibited by diphenyleneiodonium (DPI), an inhibitor of the flavin moiety of the enzyme, indicating that this group is a main source of superoxide production by the enzyme. Using measurement of [3H-L-arginine] conversion to [3H-L-citrulline], it appeared that BH4 directly increased the production of NO by NOS III. In addition, we observed that BH4 stablized the NOS III in its dimeric form, suggesting that an effect on allosteric conformation could be involved in this effect on NO production. NOS III thus appears to be a superoxide generating enzyme probably through its flavin moiety, as well as a BH4-dependent NO producing enzyme.