The activity of rifabutin against Toxoplasma gondii was investigated in vitro and in vivo. One set of in-vitro studies used either a low virulence or a high virulence T. gondii strain subcultured in drug-free conditions after exposure to various drug concentrations. No parasite growth was observed after subculture at 1 month after exposure to 6 mg/L of rifabutin for the low virulence and 12 mg/L for the high virulence strain. The IC50 of rifabutin was 26.5 mg/L in a different series of experiments using an enzyme-linked immunoassay) and the T. gondii high virulence strain. In similar experiments with clarithromycin, low virulence and high virulence parasites grew in drug-free subcultures following exposure to drug concentrations as high as 100 mg/L; the IC50 of clarithromycin exceeded 100 mg/L for the high virulence strain. In the acute toxoplasmosis murine model, mice received a 12 day treatment starting 5 days before infection with high virulence parasites. Survival was significantly improved compared with untreated controls in response to 50, 100 and 200 mg/kg/day rifabutin and 100 and 200 (but not 50) mg/kg/day clarithromycin, and with the combination of the two drugs at 25, 50 and 100 mg/kg/day. Survival was significantly improved when combination therapy was administered. The calculated ED50 values (mg/kg/day) were 160.5 for rifabutin, 119.4 for clarithromycin, and 114.8 for the combination. In the present experimental conditions rifabutin proved effective in vitro and in vivo against T. gondii and showed potentiation with clarithromycin in vivo.