Nitric oxide (NO·) is an important protective molecule in the vasculature, and endothelial NO· synthase (eNOS) is responsible for most of the vascular NO· produced. A functional eNOS oxidizes its substrate l-arginine to l-citrulline and NO·. This normal function of eNOS requires dimerization of the enzyme, the presence of the substrate l-arginine, and the essential cofactor (6R)-5,6,7,8-tetrahydro-l-biopterin (BH₄), one of the most potent naturally occurring reducing agents. Cardiovascular risk factors such as hypertension, hypercholesterolemia, diabetes mellitus, or chronic smoking stimulate the production of reactive oxygen species in the vascular wall. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidases represent major sources of this reactive oxygen species and have been found upregulated and activated in animal models of hypertension, diabetes, and sedentary lifestyle and in patients with cardiovascular risk factors. Superoxide (O₂·⁻) reacts avidly with vascular NO· to form peroxynitrite (ONOO⁻). The cofactor BH₄ is highly sensitive to oxidation by ONOO⁻. Diminished levels of BH₄ promote O₂·⁻ production by eNOS (referred to as eNOS uncoupling). This transformation of eNOS from a protective enzyme to a contributor to oxidative stress has been observed in several in vitro models, in animal models of cardiovascular diseases, and in patients with cardiovascular risk factors. In many cases, supplementation with BH₄ has been shown to correct eNOS dysfunction in animal models and patients. In addition, folic acid and infusions of vitamin C are able to restore eNOS functionality, most probably by enhancing BH₄ levels as well.