Nitric oxide (NO) is an important bio-regulatory molecule in the nervous, immune and cardiovascular systems. NO is synthesized from one of the guanidino nitrogens of L-arginine by the enzyme nitric oxide synthase (NOS). To date, several isoforms of NOS have been purified and cloned. These proteins represent a novel family of mammalian enzymes that contain both heme and cytochrome P450 reductase domains. The three prototypical forms of NOS: neuronal, cytokine-inducible and endothelial NOS, are derived from separate genes and are regulated by diverse signaling pathways. The purposes of this review are to highlight recent advances in the enzymology and molecular biology of this important family of proteins and to examine how this information pertains to the regulation of NO production.