Background. The aim of this study was to explore whether the inhibition of nuclear factor-κB (NF-κB) activation by mutant IκBα (S32,36→A) can enhance TNF-α-induced apoptosis of leukemia cells and to investigate the possible mechanism. Methods. The mutant IκBα gene was transfected into HL-60 cells by liposome-mediated techniques. G418 resistant clones stably expressing mutant IκBα were obtained by the limiting dilution method. TNF-α-induced NF-κB activation was measured by electrophoretic mobility shift assay (EMSA). The expression of bcl-XL was detected by RT-PCR and Western blot after 4 hours exposure of parental HL-60 and transfected HL-60 cells to a variety of concentrations of TNF-α. The percentage of apoptotic leukemia cells was evaluated by flow cytometry (FCM). Results. Mutant Iκ Bα protein was confirmed to exist by Western blot. The results of EMSA showed that NF-κB activation by TNF-α in HL-60 cells was induced in a dose-dependent manner, but was almost completely inhibited by mutant IκBα repressor in transfected cells. The levels of bcl-XL mRNA and protein in HL-60 cells increased after exposure to TNF-α, but changed very little in transfected HL-60 cells. The inhibition of NF-κB activation by mutant Iκ Bα enhanced TNF-α-induced apoptosis. The cytotoxic effects of TNF-α were amplified in a time- and dose-dependent manner. Conclusions. NF-κB activation plays an important role in the resistance to TNF-α-induced apoptosis. The inhibition of NF-κ B by mutant IκBα could provide a new approach that may enhance the anti-leukemia effects of TNF-α or even of other cytotoxic agents.