Background & Aims: Hepatocyte apoptosis is induced by tumor necrosis factor α (TNF-α) and Fas ligand. Although nuclear factor–κB (NF-κB) activation protects hepatocytes from TNF-α–mediated apoptosis, the NF-κB responsive genes that protect hepatocytes are unknown. Our aim was to study the role of NF-κB activation and inducible nitric oxide synthases (iNOSs) in TNF-α– and Fas-mediated apoptosis in hepatocytes. Methods: Primary cultures of hepatocytes from wild-type and iNOS knockout mice were treated with TNF-α, the Fas agonistic antibody Jo2, a nitric oxide (NO) donor (S-nitroso-N-acetylpenicillamine), an NO inhibitor (NG-methyl-L-arginine acetate), and/or adenovirus-expressing NF-κB inhibitors. Results: The IκB superrepressor and a dominant-negative form of IκB kinase beta (IKKβ) inhibited NF-κB binding activity by TNF-α or Jo2 and sensitized hepatocytes to TNF-α– and Jo2-mediated apoptosis. TNF-α and Jo2 induced iNOS messenger RNA and protein levels through the induction of NF-κB. S-nitroso-N-acetylpenicillamine inhibited Bid cleavage, the mitochondrial permeability transition, cytochrome c release, and caspase-8 and -3 activity, and reduced TNF-α– and Fas-mediated death in hepatocytes expressing IκB superrepressor. NG-methyl-L-arginine acetate partially sensitized hepatocytes to TNF-α– and Fas-mediated cell killing. TNF-α alone or Jo2 alone induced moderate cell death in hepatocytes from iNOS−/− mice. Conclusions: NO protects hepatocytes from TNF-α– and Fas-mediated apoptosis. Endogenous iNOS, which is activated by NF-κB via IKKβ, provides partial protection from apoptosis.GASTROENTEROLOGY 2001;120:1251-1262