To develop a multiantigenic vaccine against toxoplasmosis, two Toxoplasma gondii antigens, SAG1 and GRA4 selected on the basis of previous immunological and immunization studies, were chosen. We showed that DNA-based immunization with plasmids expressing GRA4 (pGRA4) or SAG1 (pSAG1mut) reduced mortality of susceptible C57BL/6 mice upon oral challenge with cysts of the 76K type II strain (62% survival). Immunization with pGRA4 and pSAG1mut, enhanced the protection (75% survival). This protection was further increased by co-inoculation with a plasmid encoding the granulocyte-macrophage colony-stimulating factor (GM-CSF) (87% survival). This latter DNA cocktail provided significant protection of less susceptible outbred Swiss OF1 mice against the development of cerebral cysts. A significantly higher survival of newborns from immunized outbred mice exposed to infection during gestation was observed (4.25+/-3.77 live pups/litter) in comparison to non-immunized mice (1.08+/-2.15 live pups/litter) without preventing parasite vertical transmission. Analysis of the immune response showed that protected animals developed a specific humoral and cellular Th1 response to native T. gondii SAG1 and GRA4 antigens. Our data demonstrated that protection was improved by associating antigens (SAG1 and GRA4) and cytokine (GM-CSF) for further development of a multiantigenic vaccine against toxoplasmosis.