Toxoplasma gondii is a ubiquitous protozoan parasite that is responsible for severe congenital birth defects and fatal toxoplasmic encephalitis in immunocompromized people. Fundamental aspects of obligate intracellular replication and pathogenesis are only now beginning to emerge for protozoan parasites. T. gondii has a fragmented pathway for salvaging pyrimidine nucleobases derived from the parasite or host cell, and this limited pyrimidine salvage capacity is funnelled exclusively through uracil phosphoribosyltransferase. Disrupting the function of this enzyme does not affect the growth of T. gondii tachyzoites, which suggests that the de novo pyrimidine biosynthesis pathway may be necessary for growth. We have examined the virulence of T. gondii mutants that lack carbamoyl phosphate synthetase II (uracil auxotrophs) to determine whether de novo pyrimidine biosynthesis is required in vivo. Here we show that T. gondii uracil auxotrophs are completely avirulent not only in immune-competent BALB/c mice but also in mice that lack interferon-gamma. A single injection of the uracil auxotroph into BALB/c mice induces long-term protective immunity to toxoplasmosis. Our findings indicate the significance of the de novo pyrimidine biosynthesis pathway for the virulence of parasitic protozoa, and suggest routes for developing vaccines and chemotherapy.