The respective pro- and antiapoptotic functions of the transcription factors p53 and nuclear factor kappaB (NF-kappaB), and their potential impact on tumorigenesis and response to tumor therapy are well recognized. The capacity of the RelA(p65) subunit of NF-kappaB to specify a pro-apoptotic outcome in response to some stimuli is less well recognized, but needs to be understood if rational manipulation of the NF-kappaB pathway is to be deployed in cancer therapy. In this report, we provide evidence that the growth-responsive nuclear protein, proenkephalin (Penk), is required, in part, for apoptosis induction, in response to activation or overexpression of p53 and RelA(p65). We describe UV-C-inducible physical associations between endogenous Penk and p53 and RelA(p65) in mammalian cell lines. Depletion of Penk by RNA interference (RNAi) substantially preserves viable cell number following exposure to UV-C irradiation or hydrogen peroxide and confers transient protection in cells exposed to the genotoxin etoposide. In virally transformed and human tumor cell lines, overexpression of nuclear Penk with overabundant or activated p53, or RelA(p65) even in the absence of p53, enhances apoptosis to the point of synergy. We have further shown that Penk depletion by RNAi substantially derepresses transcription of a range of antiapoptotic gene targets previously implicated in repression-mediated apoptosis induction by NF-kappaB and p53. Physical association of endogenous Penk with the transcriptional co-repressor histone deacetylase suggests that it may be a component of a transcriptional repression complex that contributes to a pro-apoptotic outcome, following activation of the NF-kappaB and p53 pathways, and could therefore help to facilitate an antitumor response to a broad range of agents.