Tumor necrosis factor-alpha (TNF-alpha) has been reported to activate the murine p53 promoter, which was mediated by activation and binding of the NF-kappa B transcription factor to the intact p53 consensus binding site. We show in this report that TNF-alpha at concentrations of 0.1 nM induced the DNA-binding activity of wild-type p53 fivefold in the H460a human lung carcinoma cell line. The increases in p53 DNA-binding activity by TNF-alpha in H460a cells resulted from a post-translational activation mechanism, since levels of the p53 protein remained unchanged in TNF-alpha-treated cells. Furthermore, Northern and nuclear run-off analyses ruled out an increase in transcriptional activity of the promoter of the human p53 gene in TNF-alpha-treated cells. These results are in contrast to those reported for the murine p53 gene, whose promoter is induced by TNF-alpha through activation of the NF-kappa B transcription factor. To determine the molecular basis for the lack of activation of the human p53 gene promoter by TNF-alpha, the binding of NF-kappa B to the kappa B-like sites in the murine and human p53 promoters was compared in nuclei of TNF-alpha-treated NSCLC cells by electrophoretic mobility shift assay. The human kappa B-like site differs from the murine by the substitution of two base pairs. With the exception of the H460a cell line, all NSCLC cell lines treated with 0.1 nM concentrations of TNF-alpha exhibited increased NF-kappa B DNA-binding activity. The kappa B-like site located in the murine p53 promoter bound strongly to NF-kappa B complexes in p53-negative H1299 cells. However, the kappa B-related site in the human p53 promoter showed a much lower affinity for NF-kappa B complexes. In addition, p50 homodimers specifically interacted with the human kappa B site, whereas p65/p50 heterodimers bound selectively to the mouse kappa B site. We conclude that TNF-alpha can activate human wild-type p53 in NSCLC cells by a post-translational mechanism. Moreover, the inability of TNF-alpha to activate the human p53 gene promoter, in contrast to that reported for the murine p53 gene, may be the result of differences in the interactions of the murine and human kappa B sites with NF-kappa B complexes.