The differentiation of Toxoplasma gondii tachyzoites into bradyzoites has been studied experimentally in vitro using the virulent RH strain. The differentiation was monitored by immunofluorescence detection of stage-specific proteins by monoclonal antibodies and by electron microscopy. The expression of bradyzoite-specific proteins has been induced by modifying the culture conditions in any of the following three ways: increasing the pH of the culture medium (pH 8), shifting temperature from 37 to 43 degrees C, or performing a sodium arsenite treatment. Interferon-gamma, described as involved in the control of toxoplasmosis in vivo, was inefficient to trigger bradyzoite proteins expression in HFF host cells in vitro. The pH increase and heat treatment, but not the sodium arsenite, induced the formation of cysts whose fine structure was similar to that of cysts found in the brain of mice infected by avirulent strains. Our results therefore show that the tachyzoite-bradyzoite switch is not directly dependent on an immunomodulator, but is likely to arise from an alteration of the environment of the host cell-parasite complex.