Nuclear factor-κB (NF-κB) protects hepatocytes from undergoing apoptosis during embryonic development and during liver regeneration. Activation of NF-κB is mediated through phosphorylation of its inhibitor, IκB, by a kinase complex that contains 2 IκB kinases. We analyzed the differential role of IκB kinase 1 (IKK1) and IκB kinase 2 (IKK2) in tumor necrosis factor α (TNF-α)- and interleukin-1β (IL-1β)-mediated NF-κB activation in primary rat hepatocytes. Maximal induction of IKK activity was observed 5 minutes after TNF-α and 15 minutes after IL-1β treatment, and activated IKK was able to phosphorylate GST-IκB (1-54) and GST-p65 (354-551), but not a GST-p65 (354-551) substrate with a serine-to-alanine substitution at position 536. Infection with an adenovirus containing catalytically inactive IKK2K44M (Ad5IKK2dn) completely blocked both TNF-α- and IL-1β-induced GST-IκB and GST-p65 phosphorylation, IκB degradation, and NF-κB DNA binding. Adenovirally transduced, catalytically inactive IKK1K44M (Ad5IKK1dn) reduced IKK activity and NF-κB DNA binding only slightly. Accordingly, Ad5IKK2dn induced apoptosis in 75% (±6%) of hepatocytes after 12 hours of TNF-α, which was accompanied by activation of caspases 3 and 8, nuclear fragmentation, and DNA laddering. In contrast, Ad5IKK1dn led to 21% (±2%) apoptosis in TNF-α-treated hepatocytes after 12 hours and comparatively low activity of caspases 3 and 8. Furthermore, Ad5IKK2dn completely blocked the induction of inducible nitric oxide synthase (iNOS), whereas Ad5IKK1dn had no influence on the expression of iNOS. Thus, IKK2 is the main mediator for cytokine-induced NF-κB activation in primary hepatocytes and protects against TNF-α-induced apoptosis, whereas IKK1 kinase activity is not required for NF-κB activation.