Oxidative stress has been shown to convert endothelial nitric oxide synthase (eNOS) from an NO-producing enzyme to an enzyme that generates superoxide, a process termed NOS uncoupling. This uncoupling of eNOS converts it to function as an NADPH oxidase with superoxide and hydrogen peroxide generation. eNOS uncoupling has been associated with many pathophysiologic conditions, such as heart failure, ischemia/reperfusion injury, hypertension, atherosclerosis, and diabetes. The mechanisms implicated in the uncoupling of eNOS include oxidation of the critical NOS cofactor tetrahydrobiopterin, depletion of L-arginine, and accumulation of methylarginines. All of these prior mechanisms of eNOS-derived reactive oxygen species formation occur primarily at the heme of the oxygenase domain and are blocked by heme blockers or the NOS inhibitor N-nitro-L-arginine methylester. Recently, we have identified another unique mechanism of redox regulation of eNOS through S-glutathionylation that was shown to be important in cell signaling and vascular disease. Herein, we briefly review the mechanisms of eNOS uncoupling as well as their interrelationships and the evidence for their importance in disease.