It has been shown that BH₄ ameliorates endothelial dysfunction associated with conditions such as hypertension, cigarette smoking, and diabetes. This effect has been proposed to be due to a superoxide scavenging activity of BH₄. To examine this possibility we determined the rate constant for the reaction between BH₄ and superoxide using electron paramagnetic resonance (EPR) spin trapping competition experiments with 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide (DEPMPO). We calculated a rate constant for the reaction between BH₄ and superoxide of 3.9 ± 0.2 × 10⁵ M⁻¹s⁻¹ at pH 7.4 and room temperature. This result suggests that superoxide scavenging by BH₄ is not a major reaction in vivo. HPLC product analysis showed that 7,8-BH₂ and pterin are the stable products generated from the reaction. The formation of BH₄ cation radical (BH₄^•+) was demonstrated by direct EPR only under acidic conditions. Isotopic substitution experiments demonstrated that the BH₄^•+ is mainly delocalized on the pyrazine ring of BH₄. In parallel experiments, we investigated the effect of ascorbate on 7,8-BH₂ reduction and eNOS activity. We demonstrated that ascorbate does not reduce 7,8-BH₂ to BH₄, nor does it stimulate nitric oxide release from eNOS incubated with 7,8-BH₂. In conclusion, it is likely that BH₄-dependent inhibition of superoxide formation from eNOS is the mechanism that better explains the antioxidant effects of BH₄ in the vasculature.