Apoptotic resistance of cancer cells may be overcome by the combination of treatments that activate the two major apoptotic pathways: (i) the death receptor pathway activated by death ligands and (ii) the DNA damage pathway activated by chemotherapy. We have previously shown that mesothelioma cells, resistant to most treatments, are sensitive to the combination of the death ligand tumor necrosis factor-related apoptosis inducing ligand (TRAIL/Apo2L) plus chemotherapy. We investigated a possible role for c-Jun N-terminal kinase (JNK) in the synergistic effect, knowing that JNK can be activated separately by TRAIL and by DNA damage. We chose to study the M28 and REN human mesothelioma cell lines, which are p53-inactivated, to avoid an interaction between p53 and JNK. We showed that JNK was activated by TRAIL and by etoposide and that the activation was enhanced by the combination of the two treatments. We found this activation to be caspase-independent. To inhibit the JNK pathway, we used either dominant-negative constructs of JNK1 and JNK2 (compared with dominant-negative caspase 9) or a chemical inhibitor of the JNK pathway (SP600125). In cells treated with TRAIL plus etoposide, JNK inhibition increased cell survival and decreased apoptosis significantly. In transfected M28 cells, the effect of JNK inhibition was as great as that of the dominant-negative caspase 9 construct. We conclude that JNK contributes to the synergistic effect of TRAIL combined with DNA damage by mediating signals independent of p53 leading to apoptosis.