Nuclear factor-κB (NF-κB) prevents hepatocytes from undergoing apoptosis during development and liver regeneration. Mice with inactivated glycogen synthase kinase (GSK)-3β die from hepatocyte apoptosis during development due to a defect in NF-κB activation (Hoeflich KP, Luo J, Rubie EA, Tsao MS, Jin O, and Woodgett JR. Nature 406: 86–90, 2000). In this study, we determined the role of GSK-3 in TNF-α-induced NF-κB activation and cell death in primary hepatocytes. LiCl, an established inhibitor of GSK-3, sensitized primary rat hepatocytes toward TNF-α-mediated apoptosis resulting in 90% cell death after 24 h. This was accompanied by increased caspase 8-like and 3-like activities, nuclear fragmentation and DNA laddering. LiCl treatment had no effect on IκB-α degradation, IκB kinase (IKK) activity, NF-κB binding activity, and p65 nuclear import and export, but decreased transcription of the NF-κB-dependent inducible nitric oxide synthase gene and a NF-κB-driven reporter gene. The p65 sequence revealed four potential GSK-3 phosphorylation sites within its COOH-terminal transactivation domains and recombinant GSK-3β phosphorylated glutathione S-transferase (GST)-p65(354–551), but not GST-p65(1–305) in vitro. These results indicate that GSK-3 protects hepatocytes from TNF-α-induced apoptosis through p65 phosphorylation and upregulation of NF-κB transactivation.