Augmentation of superoxide levels has been linked to impaired relaxation in hypertension, diabetes and hypercholesterolaemia. Purified endothelial nitric oxide synthase (eNOS) generates superoxide under limited availability of 5,6,7,8-tetrahydrobiopterin (BH₄). Thus alterations in endothelial BH₄ levels have been postulated to stimulate superoxide production from eNOS. This possibility was examined by determining the concentration-dependent effects of BH₄, and its analogues, on superoxide formation by eNOS. Superoxide was quantified by EPR spin trapping, which is the only available technique to quantify superoxide from eNOS. Using 5-ethoxycarbonyl-5-methyl-pyrroline N-oxide, we show that only fully reduced BH₄ diminished superoxide release from eNOS, with efficiency BH₄>6-methyl-BH₄>5-methyl-BH₄. In contrast, partially oxidized BH₄ analogues, 7,8-dihydrobiopterin (7,8-BH₂) and sepiapterin had no effect. Neither l-arginine nor N^G-nitro-l-arginine methyl ester (l-NAME) abolished superoxide formation. Together, BH₄ and l-arginine stimulated ^˙NO production at maximal rates of 148nmol/min per mg of protein. These results indicate that BH₄ acts as a ‘redox switch’, decreasing superoxide release and enhancing ^˙NO formation. This role was verified by adding 7,8-BH₂ or sepiapterin to fully active eNOS. Both 7,8-BH₂ and sepiapterin enhanced superoxide release while inhibiting ^˙NO formation. Collectively, these results indicate that the ratio between oxidized and reduced BH₄ metabolites tightly regulates superoxide formation from eNOS. The pathological significance of this scenario is discussed.