Apo-2L/TRAIL (tumor-necrosis factor-related apoptosis-inducing ligand) is a member of the tumor necrosis factor superfamily and has recently been shown to induce apoptosis through engagement of the death receptors TRAIL-R1 (DR4) and TRAIL-R2 (DR5). The transcription factor nuclear factor (NF)-kappa B regulates the expression of genes involved in cancer cell invasion, metastasis, and resistance to chemotherapy. In normal unstimulated cells, NF-kappa B is maintained in the cytoplasm with its inhibitor protein I kappa B, whereas in cancer cells, NF-kappa B is in the nucleus and constitutively activates target genes. To understand the function of NF-kappa B in TRAIL-induced apoptosis, we have analyzed the specific roles of NF-kappa B subunits. Overexpression of a transdominant-negative mutant of the inhibitory protein I kappa B alpha results in down-regulation of constitutively active NF-kappa B, induction of DR5, and tumor necrosis factor receptor (TNFR) 1-associated death domain expression and enhancement of TRAIL sensitivity. Overexpression of RelA or a transcriptional-deficient mutant of c-Rel inhibits TRAIL-induced apoptosis. Depletion of RelA in mouse embryonic fibroblasts increases cytokine-induced apoptosis, whereas depletion of c-Rel blocks this process. Overexpression of RelA subunit inhibits caspase-8 and DR4 and DR5 expression and enhances expression of cIAP1 and c-IAP2 after TRAIL treatment. By comparison, overexpression of c-Rel enhances DR4, DR5, and Bcl-X(s) and inhibits cIAP1, cIAP2, and survivin after TRAIL treatment. These results suggest that the RelA subunit acts as a survival factor by inhibiting expression of DR4/DR5 and caspase-8 and up-regulating cIAP1 and cIAP2. The dual function of NF-kappa B, as an inhibitor or activator of apoptosis, depends on the relative levels of RelA and c-Rel subunits. Thus, NF-kappa B activity may play an important role in tumor progression, and down-regulation of RelA or up-regulation of c-Rel represents a possible therapeutic target for the treatment of cancer.