Spleen cells from scid mice produce high levels of IFN-gamma when exposed to either live tachyzoites of Toxoplasma gondii or a soluble parasite extract. Small numbers of parasites are sufficient to stimulate this response, which is also induced by cell-free supernatants of cultured tachyzoites. The parasite molecules responsible for triggering IFN-gamma production are heat-labile but resistant to freezing and thawing. Depletion of NK cells or adherent cells from the splenocyte population abolishes the response. Moreover, cultured bone marrow-derived NK cells are stimulated by Toxoplasma to produce IFN-gamma, but only when supplemented with adherent peritoneal washout or thioglycollate-induced exudate cells. Supernatants of macrophages preincubated with T. gondii extract also induce IFN-gamma synthesis by cultured NK cells. Addition of neutralizing mAb against TNF-alpha abolishes the IFN-gamma response of scid spleen cells exposed to the parasite or of NK cells incubated with supernatants of adherent cells stimulated with T. gondii extract. Moreover, splenic adherent cells produce low levels of TNF-alpha in response to the parasite. Nevertheless, TNF-alpha alone is not sufficient to trigger IFN-gamma production from purified NK cell populations. These findings provide the first example of the stimulation of T-independent IFN-gamma production by a protozoan. The ability of T. gondii to trigger this pathway may underlie its induction of strong IFN-gamma-dependent nonspecific and specific cell-mediated immunity.