The endothelium plays an important role in the maintenance of vascular homeostasis. Central to this role is the endothelial production of nitric oxide (NO), synthesized by the constitutively expressed endothelial isoform of nitric oxide synthase. Vascular diseases, including hypertension, diabetes, and atherosclerosis, are characterized by impaired endothelium-derived NO bioactivity that may contribute to clinical cardiovascular events. Growing evidence indicates that impaired endothelium-derived NO bioactivity is due, in part, to excess vascular oxidative stress. This review outlines how different forms of oxidative stress can impact on NO bioactivity and discusses strategies to prevent oxidative stress-induced endothelial dysfunction.