Nitric oxide (NO) is a highly reactive free radical gas and these unique properties have been adapted for a surprising number of biological roles. In neurons, NO functions as a neurotransmitter; in immune cells, NO contributes to host defense; and in endothelial cells, NO is a major regulator of blood vessel homeostasis. In the vasculature, NO is synthesized on demand by a specific enzyme, endothelial nitric oxide synthase (eNOS) that is uniquely expressed in the endothelial cells that form the interface between the circulating blood and the various tissues of the body. NO regulates endothelial and blood vessel function via two distinct pathways, the activation of soluble guanylate cyclase and cGMP-dependent signaling and the S-nitrosylation of proteins with reactive thiols (S-nitrosylation). The chemical properties of NO also serve to reduce oxidation and regulate mitochondrial function. Reduced synthesis and/or compromised biological activity of NO precede the development of cardiovascular disease and this has generated a high level of interest in the mechanisms controlling the synthesis and fate of NO in the endothelium. The amount of NO produced results from the expression level of eNOS, which is regulated at the transcriptional and posttranscriptional levels as well as the acute posttranslational regulation of eNOS. The goal of this chapter is to highlight and integrate past and current knowledge of the mechanisms regulating eNOS expression in the endothelium and the posttranslational mechanisms regulating eNOS activity in both health and disease.