This study reports the molecular cloning and expression of an inducible nitric oxide synthase (NOS) from human hepatocytes. The cloned cDNA, named hep-NOS, shares 80% amino acid sequence homology with macrophage NOS (mac-NOS) but exhibits distinct characteristics. hep-NOS contains consensus recognition sites for FMN, FAD, and NADPH, as well as a calmodulin binding site. NOS activity in human 293 kidney cells transfected with hep-NOS cDNA is reduced by Ca2+ chelation and a calmodulin antagonist, indicating a Ca2+ dependence not observed in mac-NOS. Northern blot analysis reveals a 4.5-kb mRNA transcript in human hepatocytes and aortic smooth muscle cells following stimulation with lipopolysaccharide (LPS) and cytokines. Southern blot analysis shows that hep-NOS and endothelial NOS are distinct gene products. The findings suggest that hep-NOS is a unique form of inducible NOS, distinct from mac-NOS and other NOS isoforms, and highlights the complexity of NOS isoforms in different cell types.This study reports the molecular cloning and expression of an inducible nitric oxide synthase (NOS) from human hepatocytes. The cloned cDNA, named hep-NOS, shares 80% amino acid sequence homology with macrophage NOS (mac-NOS) but exhibits distinct characteristics. hep-NOS contains consensus recognition sites for FMN, FAD, and NADPH, as well as a calmodulin binding site. NOS activity in human 293 kidney cells transfected with hep-NOS cDNA is reduced by Ca2+ chelation and a calmodulin antagonist, indicating a Ca2+ dependence not observed in mac-NOS. Northern blot analysis reveals a 4.5-kb mRNA transcript in human hepatocytes and aortic smooth muscle cells following stimulation with lipopolysaccharide (LPS) and cytokines. Southern blot analysis shows that hep-NOS and endothelial NOS are distinct gene products. The findings suggest that hep-NOS is a unique form of inducible NOS, distinct from mac-NOS and other NOS isoforms, and highlights the complexity of NOS isoforms in different cell types.