The cell interactions that take place between Toxoplasma gondii trophozoites and the human immune system have been investigated by using an in vitro model of infection. PBMC were co-cultured with live, appropriately attenuated, trophozoites. When cells from immune (seropositive) donors were used, a proliferative response was observed. At the same time, the proliferating T cells proved capable of controlling the growth of live trophozoites. By contrast, cells from seronegative donors failed to mount a proliferative response and intracellular overgrowth of trophozoites with subsequent cell injury occurred. Actively proliferating T cells were expanded in continuous cell lines with IL-2 and periodical restimulation with Ag in the presence of autologous irradiated mononuclear cells. From some of the lines obtained, clones were also derived. Ten clones were selected for further studies. They proliferated in response to trophozoites but not to unrelated Ag. Their response required the presence of autologous monocytes-macrophages isolated from peripheral blood on Percoll density gradients. B cells that were obtained from the same donors and immortalized by EBV infection proved inefficient as APC. These data suggest that live trophozoites have to be processed by macrophages in order to be presented to T cells. Upon appropriate antigen stimulation, all of the clones produced IL-2 and IFN-gamma, a finding that was consistent with both their CD4+ surface phenotype and their helper capacity on B cell proliferation and differentiation in vitro. The supernatants of all of the stimulated clones released a factor that activated macrophages to kill intracellular trophozoites as well as an unrelated pathogen, Listeria monocytogenes. This factor was identified as IFN-gamma because it was neutralized by specific anti-IFN-gamma antibodies. The present in vitro model of response to live protozoa may prove suitable to assess the role of both T lymphocytes and macrophages in intracellular parasite infections in man. Furthermore, this experimental system may be applied to detect specific lesions of cell mediated immunity in a number of immunodeficiency syndromes.