Nitric oxide (NO), produced by endothelial NO synthase (NOS) is a key regulator of vascular tone and blood pressure. Tetrahydrobiopterin (BH4) is an essential cofactor for endothelial NOS. When BH4 levels become limiting, eNOS produces superoxide anion rather than NO and in turn further reduces NO bioavailability, resulting in endothelial dysfunction. Endothelial cell BH4 has been hypothesized to be critical in maintaining vascular function, but to date in vivo models of BH4 deficiency have achieved only partial global depletion of BH4 synthesis. We have investigated the specific role of endothelial cell BH4 using a novel line of conditional knockout mice. Mice homozygous for a floxed GCH1 (GCH1fl/fl) allele (encoding for GTPCH I protein an essential enzyme in BH4 biosynthesis) were crossed with Tie2-cre transgenic mice to produce GCH1fl/fl/Tie2-cre line, where the GCH1 gene is knocked out in endothelial cells. We have shown that the GCH1 gene is completely excised in isolated endothelial cells, with an accompanying lack of BH4 production in aortas from GCH1fl/fl/Tie2-cre mice. To assess the effects of endothelial cell BH4 deficiency on eNOS uncoupling, superoxide production in the aorta was measured using dihydroethidium fluorescence. Endothelial-derived superoxide production was significantly 4-fold greater in GCH1fl/fl/Tie2-cre mice compared to wild-type littermates, and was abolished by the NOS inhibitor L-NAME. Vasomotor studies demonstrated that GCH1fl/fl/Tie2-cre aortas had enhanced vasoconstriction to phenylephrine (Emax, 97.8±7.3 vs 118.4±5.6; P<0.05) that normalised in the presence of L-NAME. Endothelium-dependent vasodilatations in response to the receptor-mediated eNOS agonist, acetylcholine were impaired in isolated aortic rings (- log ECH50, 7.53±0.05 vs 7.30±0.05; P<0.01) and vasodilatations were abolished in the presence of L-NAME in both genotypes. The NOS-derived vasodilator in GCH1fl/fl/Tie2-cre aortas was identified as H2O2 using the scavenger PEG-catalase, which inhibited vasodilatation only in GCH1fl/fl/Tie2-cre aortas. Ex vivo supplementation of aortic rings with BH4 analogue sepiapterin restored normal endothelial function and abolished eNOS-derived H2O2 production in GCH1fl/fl/Tie2-cre aortas. Non-invasive blood pressure measurement using tail-cuff plethysmography demonstrated higher systolic blood pressure in GCH1fl/fl/Tie2-cre mice than wild-type littermates (96.4±0.8 in WT vs 103.4±1.3 in GCH1fl/fl/Tie2-cre) that normalised when L-NAME was given in drinking water (114.3±1.1 in WT vs 113.4±1.8 in GCH1fl/fl/Tie2-cre). These findings demonstrate that endothelial cell BH4 plays a pivotal role in maintaining normal vascular function and determining the formation of the alternative eNOS-derived vasodilators, NO vs H2O2.