The transcription factor nuclear factor-kappaB (NF-kappaB) plays a central role in stress-induced transcriptional activation and has been implicated in chemoresistance of cancers. In the present study, we investigated the role of NF-kappaB in inducible chemoresistance of neuroblastoma. Doxorubicin, VP16 and the cytotoxic ligand TRAIL trigger NF-kappaB activation, whereas cisplatin and taxol have no impact on NF-kappaB activity. Specific inhibition of NF-kappaB activation by overexpression of dominant-negative mutant IkappaBalpha-super-repressor does not alter cell death upon doxorubicin or VP16 treatment, although it prevents doxorubicin- or VP16-mediated NF-kappaB activation. By comparison, inhibition of TRAIL-stimulated NF-kappaB activation by IkappaBalpha-superrepressor or the small molecule NF-kappaB inhibitor BMS-345541 significantly enhances TRAIL-induced apoptosis, pointing to an antiapoptotic function of NF-kappaB in TRAIL-mediated apoptosis. Analysis of signaling pathways reveals that NF-kappaB inhibition prevents TRAIL-triggered up-regulation of Mcl-1, promoting TRAIL-induced cytochrome c release and activation of caspases. Accordingly, knockdown of Mcl-1 by RNA interference significantly enhances TRAIL-induced apoptosis and also increases sensitivity of neuroblastoma cells to CD95- or chemotherapy-induced apoptosis. In conclusion, NF-kappaB regulates apoptosis in a stimulus-specific manner in neuroblastoma cells and confers protection against TRAIL-induced apoptosis. By demonstrating that NF-kappaB inhibition sensitizes neuroblastoma cells for TRAIL-induced apoptosis, our findings have important implications. Thus, NF-kappaB inhibitors may open new perspectives to potentiate the efficacy of TRAIL-based protocols in the treatment of neuroblastoma.