Tumor necrosis factor (TNF) signaling leads to pleiotropic responses in a wide range of cell types, in part by activating antiapoptotic and proapoptotic signaling pathways. Thus, although TNF can cause apoptosis and may prove useful in the treatment of malignancies, most cells are resistant to TNF-induced cell death unless de novo protein synthesis is inhibited. Previous studies suggested that TNF activation of the nuclear factor (NF)-kappaB transcription factor family antagonizes the proapoptotic signals initiated by TNF-alpha. TNF receptor-associated factor (TRAF)2 has also been shown to mediate crucial antiapoptotic signals during TNF stimulation, yet is not essential in activation of NF-kappaB under physiologic conditions, thus raising questions about the relationship between these antiapoptotic pathways. We report here that inhibition of TRAF2 and NF-kappaB function in primary cells, by coexpression of a constitutive repressor of multiple NF-kappaB/Rel proteins (IkappaBalpha.DN) and a dominant negative form of TRAF2 (TRAF2.DN), synergistically enhanced TNF-induced apoptosis. The effects were stimulus dependent, such that neither inhibitory molecule affected Fas- and daunorubicin-induced apoptosis to the same degree as TNF-induced death. These findings indicate that the NF-kappaB and TRAF2 pathways activate independent antiapoptotic mechanisms which act in concert to suppress the proapoptotic signals induced by TNF-alpha.