Reduced glutathione (GSH) plays a crucial role in hepatocyte function, and GSH depletion by diethyl maleate was shown previously to inhibit expression of NF-kappaB target genes induced by tumor necrosis factor alpha (TNFalpha) and sensitize primary cultured mouse hepatocytes to TNF-mediated apoptotic killing. Here we demonstrate in the same system that GSH depletion down-regulates TNF-induced NF-kappaB transactivation via two mechanisms, depending on the extent of the depletion. With moderate GSH depletion (approximately 50%), the down-regulation is IkappaB kinase (IKK)-independent and likely acts on NF-kappaB transcriptional activity because TNF-induced IKK activation, IkappaBalpha phosphorylation and degradation, NF-kappaB nuclear translocation, NF-kappaB DNA binding in vitro, and NF-kappaB subunit RelA(p65) recruitment to kappaB sites of target gene promoters all appear unaltered. On the other hand, with profound GSH depletion (approximately 80%), the down-regulation also is IKK-dependent, and a timeline is established linking the inhibition of polyubiquitination of receptor-interacting protein 1 in TNF receptor 1 complex to partial blockage of IKK activation, IkappaBalpha phosphorylation and degradation, and NF-kappaB nuclear translocation. Of note, pretreatment with antioxidant trolox protects against the inhibitory effect of profound GSH depletion on IKK activation and NF-kappaB nuclear translocation but fails to restore expression of NF-kappaB target genes, revealing both IKK-dependent and -independent inhibition. These findings provide new insights into the complex effects of oxidative stress and redox perturbations on the NF-kappaB pathway.