Immunostaining and EMSA revealed that NF-κB was activated strongly by TNF/IFN-α compared to TNF alone in a human colon adenocarcinoma cell line, RPMI4788. Although inhibition of activated NF-κB, by using an NF-κB decoy, reduced cell viability after treatment with TNF only, NF-κB decoy resulted in recovery of cell viability after TNF/IFN-α treatment. Caspase-3 activity was increased in cells induced by TNF/IFN-α, while suppression of caspase-3 activity was observed in cells transfected with NF-κB decoy and then treated by TNF/IFN-α. On the other hand, Fas expression was strongly enhanced by TNF/IFN-α, and inhibition of TNF/IFN-α-induced NF-κB activation, by using NF-κB decoy, decreased Fas expression. Cell viability and caspase-3 activity decreased in cells treated with TNF/IFN-α and anti-FasL antibody. Taken together, our findings suggest that activated NF-κB induced by the crosstalk between TNF and IFN-α is a novel pro-apoptotic signal acting via enhancement of Fas expression.