To investigate early immune responses to the intracellular parasite Toxoplasma gondii, we examined the capacity of nonimmune splenocytes to respond in vitro to intact tachyzoites and soluble tachyzoite antigen (Ag). Both types of stimuli induced high levels of proliferation as well as interferon gamma (IFN-gamma) secretion. Based on several key criteria, the response appeared to be driven by a superantigen present in the parasite. Thus, stimulation of C57BL/6 spleen cells with T. gondii resulted in a preferential threefold expansion of a T cell population expressing the V beta 5 chain of the T cell receptor, and a survey of different inbred mouse strains revealed an inverse correlation between Ag-induced proliferation and genetic deletion of V beta 5. Moreover, proliferation was induced using irradiated Ag-pulsed and infected splenic adherent cells, and was blocked by a major histocompatibility complex class II-specific monoclonal antibody. Furthermore, paraformaldehyde-fixed IAb-, IAk-, and IEk-transfected fibroblast lines were able to specifically bind T. gondii Ag and drive proliferation of T lymphocytes, demonstrating that the response can be mediated by allogeneic class II molecules, and that it does not require cellular Ag processing. It is interesting to note that after 1 wk of culture with Ag, up to 70% of the expanded V beta 5-expressing cells were CD8+. These results provide the first description of a superantigen activity in a protozoan pathogen. In the case of T. gondii, superantigen-driven expansion of IFN-gamma-secreting CD8+ lymphocytes may play a role in the development of the dominant IFN-gamma-dependent, cell-mediated immunity characteristic of infection with this parasite.