Elevated endogenous JNK activity and resistance to Fas receptor-mediated apoptosis have recently been implicated in progression of prostate cancer and can promote resistance to apoptosis in response to chemotherapeutic drugs. In addition, JNK has been demonstrated to promote transformation of epithelial cells by increasing both proliferation and survival. Although numerous studies have reported a role for JNK in promoting Fas receptor-mediated apoptosis, there is a paucity in the literature studying the antiapoptotic function of JNK during Fas receptor-mediated apoptosis. Consequently, we have used the recently described specific JNK inhibitor SP600125 and RNA interference to inhibit endogenous JNK activity in the prostate carcinoma cell line DU 145. We demonstrated that endogenous JNK activity increased the expression of a kinase, HIPK3, that has previously been implicated in multidrug resistance in a number of tumors. HIPK3 has also been reported to phosphorylate FADD. The interaction between FADD and caspase-8 was inhibited, but abrogation of JNK activity or HIPK3 expression was found to restore this interaction and increased the sensitivity of DU 145 cells to Fas receptor-mediated apoptosis. In conclusion, we present novel evidence that JNK regulates the expression of HIPK3 in prostate cancer cells, and this contributes to increased resistance to Fas receptor-mediated apoptosis by reducing the interaction between FADD and caspase-8.