Nitric oxide (NO), endogenously synthesized by inducible NO synthase (iNOS), serves antioxidant and antiapoptotic functions in settings characterized by oxidative stress and proinflammatory cytokines such as sepsis and shock. However, the redox-sensitive mechanisms regulating hepatocyte expression of iNOS are largely unknown. In interleukin-1beta (IL-1beta)-stimulated hepatocytes exposed to superoxide, we demonstrate that hepatocyte nuclear factor-4alpha (HNF-4alpha) acts as an activator of redox-associated hepatocyte iNOS expression at the level of protein, mRNA, and promoter activation. In the absence of HNF-4alpha, this redox-mediated enhancement is ablated. HNF-4alpha functional activity is associated with a unique serine/threonine kinase-mediated phosphorylation pattern. This suggests that a redox-sensitive kinase pathway targets HNF-4alpha to augment hepatocyte iNOS expression. Previous studies have not addressed a redox-dependent kinase signaling pathway that targets HNF-4alpha and enhances hepatocyte iNOS gene transcription. A unique pattern of phosphorylation determines HNF-4alpha activity as a trans-activator of IL-1beta-mediated hepatocyte iNOS expression in the presence of oxidative stress.