This study reports the molecular cloning and expression of an inducible nitric oxide synthase (NOS) from human hepatocytes, termed hep-NOS. The hep-NOS cDNA was cloned from human hepatocytes stimulated with lipopolysaccharide (LPS) and cytokines, including tumor necrosis factor (TNF), interleukin 1 (IL-1), and interferon γ (IFN-γ). The hep-NOS cDNA showed 80% amino acid sequence homology to macrophage NOS (mac-NOS). Like other NOS isoforms, hep-NOS contains recognition sites for FMN, FAD, and NADPH, as well as a consensus calmodulin binding site. However, unlike mac-NOS, hep-NOS activity is Ca²+ dependent, which was confirmed by experiments using Ca²+ chelation and a calmodulin antagonist. Northern blot analysis revealed a 4.5-kb mRNA in both human hepatocytes and aortic smooth muscle cells following stimulation with LPS and cytokines. Southern blot analysis showed that hep-NOS and endothelial NOS (eNOS) have distinct genomic restriction enzyme fragments, suggesting they are distinct gene products. The study also demonstrated that NOS activity is inducible in human hepatocytes and smooth muscle cells, with hep-NOS mRNA levels closely matching NOS activity. The results indicate that hep-NOS is an inducible form of NOS that is distinct from mac-NOS, brain NOS, and eNOS. The study provides evidence that human hepatocytes express an inducible NOS, which may play a role in immune responses and inflammatory processes. The findings contribute to the understanding of NOS isoforms and their roles in various physiological and pathological conditions.This study reports the molecular cloning and expression of an inducible nitric oxide synthase (NOS) from human hepatocytes, termed hep-NOS. The hep-NOS cDNA was cloned from human hepatocytes stimulated with lipopolysaccharide (LPS) and cytokines, including tumor necrosis factor (TNF), interleukin 1 (IL-1), and interferon γ (IFN-γ). The hep-NOS cDNA showed 80% amino acid sequence homology to macrophage NOS (mac-NOS). Like other NOS isoforms, hep-NOS contains recognition sites for FMN, FAD, and NADPH, as well as a consensus calmodulin binding site. However, unlike mac-NOS, hep-NOS activity is Ca²+ dependent, which was confirmed by experiments using Ca²+ chelation and a calmodulin antagonist. Northern blot analysis revealed a 4.5-kb mRNA in both human hepatocytes and aortic smooth muscle cells following stimulation with LPS and cytokines. Southern blot analysis showed that hep-NOS and endothelial NOS (eNOS) have distinct genomic restriction enzyme fragments, suggesting they are distinct gene products. The study also demonstrated that NOS activity is inducible in human hepatocytes and smooth muscle cells, with hep-NOS mRNA levels closely matching NOS activity. The results indicate that hep-NOS is an inducible form of NOS that is distinct from mac-NOS, brain NOS, and eNOS. The study provides evidence that human hepatocytes express an inducible NOS, which may play a role in immune responses and inflammatory processes. The findings contribute to the understanding of NOS isoforms and their roles in various physiological and pathological conditions.