Tumor necrosis factor alpha (TNFα) generates a potent cytotoxic effect, however many cancer cells are resistant to TNFα-Mediated Killing and the cause of the diferential sensitivity remains to be elucidated. In this study, we demonstrated that TNFα induced cell death in four different human colon cancer cell lines. The degree of cytotoxic effect was different in each cell line, in that HCT-15 was relatively sensitive, while DLD-1, HT-29 and WiDr were relatively resistant. TNFα induced apoptotic changes such as morphological changes, DNA fragmentation and activation of caspase-3 in HCT-15, but to a lesser degree in the others. Transcriptional expression of TNFR1(p55), as well as that of FLICE, Fas, FADD, DR3, FAF, TRADD, and RIP was similar in these cell lines, indicating that the susceptibility to TNFα-induced apoptosis may not be determined by the constitutive expression level of these factors. Interestingly, the cytotoxic effect of TNFα was well correlated with the DNA binding activity of NF-κB in the colon cancer cell lines. Further, the overexpression of a non-phosphorylated mutant form of IκBα enhanced the cytotoxicity of TNFα in the resistant cell line, DLD-1, indicating that NF-κB activity may determine the sensitivity of colon cancer cells to TNFα-induced apoptosis. Thus, our results indicate that modulation of NF-κB activity may provide a useful tool to sensitize colon cancer cells to TNFα treatment.