Nitric oxide (NO), a diffusible free radical gas, acts as a neurotransmitter in brain and peripheral nervous system. It accounts for the activity of endothelium-derived relaxing factors, which stimulate vasodilatation by releasing NO from the endothelium. NO is synthesized by L-arginine, oxygen, and NADPH by three known isoforms of heme-containing flavoproteins termed NO synthase (NOS, I-III, mol wt. ~130-160kD). One group of enzyme is constitutive, agonist-triggered, and dependent on Ca2+/Calmodulin and is inhibited by L- arginine analogues (L-NNA, L-NMMA). It is found in endothelium, adrenal glands, brain and platelets. The other principle group is inducible, Ca2+/Calmodulin-independent, and inhibited by NMMA and L-NNA. It has been found in macrophage, hepatocytes, tumor cells, vascular smooth muscle and endothelial cells. Analyses of cDNA clones have identified three distinct NOS genes in mammals: neuronal (nNOS/bNOS/NOS-I), endothelial (eNOS/NOS-III), and macrophage (mNOS/iNOS/NOS-II). Both nNOS and eNOS are constitutive and the mNOS/iNOS is inducible. Sequence homology among different cloned isoforms is ~50%. Human, rat, mouse, and bovine eNOS/NOS-3 are ~1202-1205aa proteins.