Objective-6R-5,6,7,8-tetrahydro-L-biopterin (6R-BH4) is a cofactor for endothelial nitric oxide synthase but also has antioxidant properties. Its stereo-isomer 6,7, and structurally similar pterin 6R, 6,7, are also antioxidants but have no cofactor function. When endothelial nitric oxide synthase is 6R-BH4 -deplete, it synthesizes superoxide rather than nitric oxide. Reduced nitric oxide bioavailability by interaction with reactive oxygen species is implicated in endothelial dysfunction (ED). 6R-BH4 corrects ED in animal models of ischemia reperfusion injury (IRI) and in patients with cardiovascular risks. It is uncertain whether the effect of exogenous 6R-BH4 on ED is through its cofactor or antioxidant action. Methods and Results-In healthy volunteers, forearm blood flow was measured by venous occlusion plethysmography during intra-arterial infusion of the endothelium-dependent vasodilator acetylcholine, or the endothelium-independent vasodilator glyceryl trinitrate, before and after IRI. IRI reduced plasma total antioxidant status (Pϭ0.03) and impaired vasodilatation to acetylcholine (Pϭ0.01), but not to glyceryl trinitrate (Pϭ0.3). Intra-arterial infusion of 6R-BH4, 6S-BH4 and NH4 at approximately equimolar concentrations prevented IRI. Conclusion-IRI causes ED associated with increased oxidative stress that is prevented by 6R-BH4, 6S-BH4, and NH4, an effect mediated perhaps by an antioxidant rather than cofactor function. R-5,6,7,8-terahydro-L-biopterin (6R-BH4) is an essential cofactor for endothelial nitric oxide synthase (NOS) that catalyzes production of the vasodilator and atheroprotective mediator NO. 1,2 Biosynthesis of 6R-BH4 occurs in the endothelium, promoting NO synthesis by facilitating electron transfer from the reductase domain of NOS to arginine. 6R-BH4 also stabilizes the NOS dimer and exerts an allosteric effect, enhancing substrate binding. 3 Changes in 6R-BH4 availability influence NO production. A reduction in 6R-BH4 leads not only to a diminished NO synthesis but also to the generation of NOS-derived superoxide (O 2 ⅐Ϫ ) that has been implicated in the development of endothelial dysfunction. 4 Ischemia reperfusion injury (IRI), such as occurs after thrombolysis or balloon angioplasty, is associated with endothelial dysfunction, which may contribute to cellular damage. The mechanism underlying endothelial dysfunction in IRI is incompletely understood but adhesion of activated neutrophils to endothelial cells, 5 an increase in oxygen radical generation, 6,7 the elaboration of inflammatory cytokines, and a reduction in NO production 8,9 are all believed to play a role.In humans, local delivery of 6R-BH4 by intra-arterial infusion improves endothelium dependent vasodilation in patients with coronary artery disease, 10 type II diabetes mellitus, 11 elevated cholesterol, 12 raised blood pressure, 13 and in smokers, 14 leading to the proposal that an acquired deficiency in 6R-BH4 and defective NOS catalysis underlies endothelial dysfunction in these states. Supplementation with 6R-BH4 also red...