The c‐Jun NH₂‐terminal kinase (JNK) is involved in the regulation of cell death, but its role in tumor necrosis factor (TNF)‐α‐and Fas‐mediated apoptosis in primary cells is not well defined. In primary rat hepatocytes expressing an IκB superrepressor, the JNK inhibitor SP600125 strongly decreased TNF‐α‐induced cell death, caspase 3 activation, and DNA laddering. In contrast, SP600125 did not rescue mouse hepatocytes from Fas‐induced apoptosis. Apoptosis in mouse hepatocytes, induced by human TNF‐α, was blocked by SP600125, indicating that TNF‐receptor (TNF‐R) 1‐mediated JNK activation is important for TNF‐α‐induced death. However, mouse TNF‐α was more efficient than human TNF‐α in activating JNK and killing mouse hepatocytes, suggesting that TNF‐R1 and TNF‐R2 cooperate in JNK activation and apoptosis. SP600125 rescued actinomycin D‐pretreated hepatocytes and hepatocytes expressing a dominant negative c‐Jun from TNF‐α, indicating that JNK exerts its proapoptotic effect independently of transcription and c‐Jun. SP600125 delayed the mitochondrial permeability transition, inhibited cytochrome c release and prevented bid degradation after TNF‐α, suggesting that JNK‐regulated proapoptotic factors act upstream of the mitochondria. Moreover, overexpression of JNK1 activated a mitochondrial death pathway in hepatocytes, albeit less efficiently than TNF‐α . This study demonstrates that JNK augments TNF‐α‐induced apoptosis in hepatocytes through a signaling pathway that is distinct from the pathway by which it regulates proliferation.