Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) induces apoptosis in a wide range of malignant cells. However, several cancers, including human hepatoma, are resistant to TRAIL. In this study, we analyzed TRAIL-induced pro- and antiapoptotic signaling pathways in human hepatoma cells. Nuclear factor k B (NF-kB) was found to be a critical TRAIL-induced antiapoptotic factor in the PLC/PRF/5, HepG2, and Hep3B cell lines. TRAIL-induced NF-kB activation was preceded by IkBα kinase (IKK) activation and IkBα degradation and depended on TRAF2, NF-kB-inducing kinase (NIK), IKK1, and IKK2. Accordingly, inhibition of NF-kB by adenoviral dominant negative (dn) TRAF2, NIKdn, IKK1 dn, IKK2dn, or IkBsr sensitized PLC/PRF/5 cells to rhTRAIL, resulting in 40% to 50% cell death after 48 hours as compared with < 10% with rhTRAIL alone. Agonistic anti-TRAIL receptor 1 and anti-TRAIL receptor 2 antibodies or combinations of both were equally efficient in inducing apoptosis as rh TRAIL, indicating that decoy receptors did not contribute to resistance toward TRAIL under the conditions of our study. TRAIL-mediated apoptosis depended on FADD, caspase 8 and 3 as demonstrated by the ability of FADDdn, CrmA, and pharmacologic caspase inhibitors to prevent apoptosis. Confocal microscopy showed the onset of the mitochondrial permeability transition (MPT) 5 hours after rh TRAIL plus actinomycin D, which was followed by cytochrome c release. The MPT was critical for TRAIL-induced apoptosis as demonstrated by the ability of pharmacologic MPT inhibitors to completely protect PLC/PRF/5 cells. In conclusion, NF-kB prevents TRAIL-induced apoptosis in human hepatoma through a TRAIL-activated TRAF2-NIK-IKK pathway. Inhibition of NF-kB unmasks a TRAIL-induced apoptotic signaling cascade that involves FADD, caspase 8, the MPT, and caspase 3. (HEPATOLOGY2002;36:1498–1508).