The in vitro effects of three synthetic, cis-fused, bicyclic 1,2,4-trioxanes (pentatroxane, thiahexatroxane, and hexatroxanone) against intracellular Toxoplasma gondii were studied. Unactivated peritoneal macrophages were infected with the virulent RH strain of T. gondii and exposed to the 1,2,4-trioxanes at different concentrations. The antitoxoplasmic activity of the drugs was first assessed with [3H]uracil, which is incorporated by the parasite but not the host cell. Pentatroxane and thiahexatroxane were the most active, exhibiting 90% inhibitory concentrations of 6.8 and 5.3 ,g/ml, respectively. Furthermore, microscopic examination of the infected macrophages after treatment with pentatroxane, thiahexatroxane, and hexatroxanone at their respective 90% inhibitory concentrations confirmed the inhibition of intracellular growth of T. gondii. Their activities were comparable to those of pyrimethamine (1 ,ug/ml) and pyrimethamine (0.1 ,ig/ml) in combination with sulfadiazine (25 ,ug/ml). Pentatroxane and thiahexatroxane were also able to inhibit intracellular growth of T. gondii within nonprofessional phagocytes (HeLa cells), suggesting that the antitoxoplasmic activity was not caused by a macrophage-specific mechanism, such as macrophage activation. However, the 1,2,4-trioxanes were not active against extracellular T. gondii. Pentatroxane and thiahexatroxane are new, promising compounds that deserve further study to assess their usefulness for treating toxoplasmosis.