The plasma level of NO_x, i.e., the sum of NO₂⁻ and NO₃⁻, is frequently used to assess NO bioavailability in vivo. However, little is known about the kinetics of NO conversion to these metabolites under physiological conditions. Moreover, plasma nitrite recently has been proposed to represent a delivery source for intravascular NO. We therefore sought to investigate in humans whether changes in NO_x concentration are a reliable marker for endothelial NO production and whether physiological concentrations of nitrite are vasoactive. NO₂⁻ and NO₃⁻ concentrations were measured in blood sampled from the antecubital vein and brachial artery of 24 healthy volunteers. No significant arterial-venous gradient was observed for either NO₂⁻ or NO₃⁻. Endothelial NO synthase (eNOS) stimulation with acetylcholine (1–10 μg/min) dose-dependently augmented venous NO₂⁻ levels by maximally 71%. This effect was paralleled by an almost 4-fold increase in forearm blood flow (FBF), whereas an equieffective dose of papaverine produced no change in venous NO₂⁻. Intraarterial infusion of NO₂⁻ had no effect on FBF. NOS inhibition (N^G-monomethyl-l-arginine; 4–12 μmol/min) dose-dependently reduced basal NO₂⁻ and FBF and blunted acetylcholine-induced vasodilation and NO release by more than 80% and 90%, respectively. In contrast, venous NO₃⁻ and total NO_x remained unchanged as did systemic arterial NO₂⁻ and NO₃⁻ levels during all these interventions. FBF and NO release showed a positive association (r = 0.85; P < 0.001). These results contradict the current paradigm that plasma NO₃⁻ and/or total NO_x are generally useful markers of endogenous NO production and demonstrate that only NO₂⁻ reflects acute changes in regional eNOS activity. Our results further demonstrate that physiological levels of nitrite are vasodilator-inactive.