Tumor necrosis factor (TNF), first described as a cytokine with tumor-necrotizing activity, is now known to be a pleiotropic molecule. The molecular mechanisms responsible for the cytotoxic activity of TNF on malignant cells are still largely unknown. In this study, we report that the protein kinase inhibitor staurosporine (56 to 1500 nM) increases about 500 times the in vitro cytotoxic activity of TNF for several murine and human tumor cell lines. Even some tumor cell lines which are resistant to TNF cytotoxicity could be sensitized to TNF killing by staurosporine. In the L929 fibrosarcoma cell line, staurosporine also enhanced the transcriptional activation of interleukin 6 synthesis by TNF (500-fold stimulation at 56 nM). At the biochemical level, staurosporine increased the TNF-mediated activation of phospholipases C and D and the transcription factor NF-kappa B in L929 cells. The TNF-sensitizing effect of staurosporine does not seem to be mediated by one of the currently known staurosporine-sensitive kinases, as various other inhibitors which also inhibit one or more of these kinases were not synergistic with TNF. Interestingly, staurosporine (1 microgram) also enhanced the in vivo antitumor activity of TNF against a murine tumor model (L929 fibrosarcoma) in athymic nude mice (Swiss-nu/nu; s.c. treatment). These results suggest that TNF responsiveness of tumor cells is regulated by a novel staurosporine-sensitive target and that the combination of TNF and staurosporine may open new strategies of tumor treatment.