Tumor necrosis factor (TNF)-α has been shown to exert cytotoxic or cytostatic effects on tumor cells, but susceptibility to TNF-α varies among different types of cells. TNF-α activates a transcription factor, nuclear factor-κB (NF-κB), which induces a wide variety of genes and causes pleiotrophic responses. In this study, the relationship between susceptibility to TNF-α and activation of NF-κB was investigated in six human malignant glioma cell lines. Cell proliferation analysis revealed that only one cell line, SK-MG-1, was sensitive to TNF-α and that the other five, including U-251MG, were resistant. Electrophoretic mobility-shift assay showed that TNF-α strongly activated a subtype of NF-κB, the p50-p65 heterodimer, in all of the resistant cell lines tested. However, this activation was weak in the sensitive cell line, SK-MG-1. Activation of NF-κB by TNF-α in the resistant cell lines resulted in a significant increase of a reporter gene expression driven by NF-κB site, suggesting a possibility that activation of p50-p65 confers resistance to TNF-α. To test this hypothesis, we established a stable cell line that expresses an inducible dominant negative NF-κB (p65 DN) protein in one of the TNF-α-resistant cell lines, U-251MG. In the established clone, induction of p65 DN protein decreased TNF-α-dependent increase in the DNA binding of p50-p65 heterodimer and NF-κB-dependent reporter gene activity. Although no growth inhibition of this clone was observed by TNF-α treatment, induction of p65 DN together with TNF-α resulted in a significant decrease in cell number. Cell cycle analysis revealed that this growth inhibition was due to the impairment of cell cycle progression. These results indicate that an active NF-κB complex, such as the p50-p65 heterodimer, plays a crucial role in the progression of cell cycle in malignant glioma cells. Refractoriness to TNF-α treatment could be prevented by inhibiting NF-κB activation.