Development of prophylactic vaccines against Toxoplasma gondii is based on the observation that latently infected subjects are protected against secondary infection during pregnancy. Cocktail DNA vaccines have been shown to provide high resistance to parasite challenge, and latently infected mice are protected against acute disease. In order to characterize the associated Th1 cellular immune responses in vivo, we used H2-K(k) bone marrow macrophage cell lines constitutively expressing T. gondii GRA1, GRA7 or ROP2 antigens, for the in vivo characterization of antigen-specific T cells in an antigenic challenge model, and as target cells in an in vivo CTL assay. In latently infected C3H/HeN mice, CD4(+) and CD8(+) T cells were recruited to the peritoneal cavity after i.p. challenge with these syngeneic cell lines. GRA1 and GRA7-specific T cells from infected mice were IFN-gamma(+) FasL(-) CD107(-). No IFN-gamma or lytic markers were observed against ROP2. In cocktail DNA vaccinated C3H/HeN mice, the response was restricted to GRA1-specific CD8(+) IFN-gamma(-) FasL(-) CD107(+) T cells. Target cells expressing GRA1 and GRA7, but not ROP2, were efficiently killed in an in vivo CTL assay in latently infected mice, while in DNA vaccinated mice only lysis of GRA1 expressing target cells was observed. Both forms of immunization, DNA vaccination and latent infection, completely protected mice against acute Toxoplasmosis. The results obtained in this work suggest that distinct in vivo cytolytic effector mechanisms are at work in DNA vaccinated and latently infected mice, but both converge to protect against acute toxoplasmosis.