Nitric oxide (NO) is a potent biologic mediator with diverse physiologic and pathophysiologic roles. NO is produced from L-arginine by the family of nitric oxide synthase (NOS) enzymes, forming the free radical NO and citrulline as byproduct. Three distinct isoforms of the NOS enzyme have been isolated and represent the products of three different genes. Two of the NOS enzymes are continuously present and are termed constitutive NOS (cNOS). One cNOS enzyme was identified in neurons, and the other in endothelial cells. The two cNOS enzymes are contrasted with the third NOS isoform, inducible NOS, which is not typically expressed in resting cells and must first be induced by certain cytokines, microbial products, or lipopolysaccharide. Since NO production has both beneficial and detrimental consequences, understanding the molecular mechanisms that regulate NOS expression is critical to the control of NO release in homeostatic and pathophysiologic conditions. The purpose of this review is to describe the molecular biology of NO synthases, with particular emphasis on the regulation of the human NO synthase genes. Transcriptional and post-transcriptional regulation of neuronal and endothelial cNOS genes will be reviewed first, followed by the molecular regulation of the inducible NOS gene.