BALB/c mice were intramuscularly immunized with low doses (25–50 g) of DNA cocktail containing plasmids encoding the full-length SAG1/P30 and the 196–561 terminal sequence of ROP2 genes. This immunization resulted in a Th1-type response with predominance of IgG2a and a specific T-cell proliferation with high levels of interferon-gamma (IFN-) secretion, whereas no IL-4 was detected. Moreover, DNA cocktail immunization induced a long-lasting protection against a lethal challenge with the highly virulent Toxoplasma gondii RH strain, whereas low doses of single genes were not protective. These results support further investigations to achieve a multigene anti-T. gondii DNA vaccine.