Hepatocyte resistance to tumor necrosis factor α (TNF)-induced apoptosis is dependent on activation of the transcription factor nuclear factor κB (NF-κB). To determine the mechanism by which NF-κB protects against TNF toxicity, the effect of NF-κB inactivation on the proapoptotic c-Jun NH2-terminal kinase (JNK) signaling pathway was examined in the rat hepatocyte cell line RALA255-10G. Adenovirus-mediated NF-κB inactivation led to a prolonged activation of JNK and increased activating protein-1 (AP-1) transcriptional activity in response to TNF treatment. Inhibition of the function of the JNK substrate and AP-1 subunit c-Jun blocked cell death from NF-κB inactivation and TNF as determined by measures of cell survival, numbers of apoptotic and necrotic cells, and DNA hypoploidy. Inhibition of c-Jun function blocked mitochondrial cytochrome c release and activation of caspase-3 and -7. NF-κB therefore blocks the TNF death pathway through down-regulation of JNK and c-Jun/AP-1. In conclusion, sustained JNK activation that occurs in the absence of NF-κB initiates apoptosis through a c-Jun–dependent induction of the mitochondrial death pathway.