5,6,7,8-Tetrahydrobiopterin (BH4) is an essential cofactor of nitric oxide synthases (NOSs). Oxidation of BH4, in the setting of diabetes and other chronic vasoinflammatory conditions, can cause cofactor insufficiency and uncoupling of endothelial NOS (eNOS), manifest by a switch from nitric oxide (NO) to superoxide production. Here we tested the hypothesis that eNOS uncoupling is not simply a consequence of BH4insufficiency, but rather results from a diminished ratio of BH4vs. its catalytically incompetent oxidation product, 7,8-dihydrobiopterin (BH2). In support of this hypothesis, [3H]BH4binding studies revealed that BH4and BH2bind eNOS with equal affinity ( Kd≈ 80 nM) and BH2can rapidly and efficiently replace BH4in preformed eNOS-BH4complexes. Whereas the total biopterin pool of murine endothelial cells (ECs) was unaffected by 48-h exposure to diabetic glucose levels (30 mM), BH2levels increased from undetectable to 40% of total biopterin. This BH2accumulation was associated with diminished calcium ionophore-evoked NO activity and accelerated superoxide production. Since superoxide production was suppressed by NOS inhibitor treatment, eNOS was implicated as a principal superoxide source. Importantly, BH4supplementation of ECs (in low and high glucose-containing media) revealed that calcium ionophore-evoked NO bioactivity correlates with intracellular BH4:BH2and not absolute intracellular levels of BH4. Reciprocally, superoxide production was found to negatively correlate with intracellular BH4:BH2. Hyperglycemia-associated BH4oxidation and NO insufficiency was recapitulated in vivo, in the Zucker diabetic fatty rat model of type 2 diabetes. Together, these findings implicate diminished intracellular BH4:BH2, rather than BH4depletion per se, as the molecular trigger for NO insufficiency in diabetes.