Kurloff cells are mononuclear cells possessing a large cytoplasmic inclusion body specific to the guinea pig. In this report, we present strong evidence that Kurloff cells can mediate NC activity against tumor cells in addition to their previously reported NK activity. Using an 18 h 51Cr-release assay we have shown that Kurloff cells were highly effective in killing the TNF-sensitive WEHI 164 target cell line. Lower but significant cytotoxic activity was also observed after only 4 h. However, our results suggest a different mechanism of lysis in the 4 h and 18 h assay. Lysis of WEHI 164 target cells by Kurloff cells in the 4 h assay could be strongly increased in the presence of TPA alone or in combination with ionomycin whereas ionomycin alone was uneffective. In contrast, stimulation of Kurloff cells for 18 h with ionomycin alone or in combination with TPA could induce the release of TNF-like factor(s) as observed by the TNF bioassay using L-929 TNF-sensitive target cells. Release of TNF-like factor(s) could also be induced by stimulation with WEHI 164 target cells. Supernatants of Kurloff cells stimulated for 18 h with TPA + ionomycin were also highly cytotoxic against WEHI 164 target cells, but not against the TNF-resistant P815 target cell line. Pretreatment of these supernatants with antimurine TNF alpha antibodies could almost completely inhibit their cytotoxic activity against WEHI 164 target cells. In contrast, supernatants of Kurloff cells stimulated for only 4 h did not show any TNF-like activity against the L-929 target cell line and were not cytotoxic against WEHI 164 target cells even after 18 h. Taken together, these results suggest that Kurloff cells can mediate NC activity against tumor cells in addition to their previously reported NK activity. By using multiple lytic pathways, these cells may play a crucial role in anti-tumor surveillance and defenses.