Toxoplasma gondii is an obligate intracellular parasite that is a common opportunistic pathogen of the central nervous system in AIDS patients. Gamma interferon (IFN-γ) alone or in combination with interleukin-1 (IL-1), IL-6, or tumor necrosis factor alpha significantly inhibits the growth of T. gondii in murine astrocytes, suggesting these are important nonimmune effector cells in the brain. Inhibition was found to be independent of a nitric oxide-mediated or tryptophan starvation mechanism. Both reactive oxygen intermediates and iron deprivation are IFN-γ-mediated mechanisms known to operate against intracellular parasites in other cell types. Astrocytes generated from mice genetically deficient in the production of reactive oxygen intermediates (phox^−/−mice) were found to inhibit growth of T. gondii when stimulated with IFN-γ alone or in combination with other cytokines. The reactive oxygen inhibitor catalase and the reactive oxygen scavengers mannitol and thiourea failed to reverse the IFN-γ-induced inhibition of T. gondii in astrocytes. These data indicate that IFN-γ-induced inhibition in astrocytes is independent of reactive oxygen intermediates. IFN-γ-induced inhibition could not be reversed by the addition of iron salts, ferric citrate, ferric nitrate, or ferric transferrin. Pretreatment of astrocytes with desferrioxamine also did not induce the inhibition of T. gondii. These data indicate that the mechanism of IFN-γ inhibition was not due to iron deprivation. IFN-γ had no effect on T. gondii invasion of astrocytes, but inhibition of growth and loss of tachyzoite vacuoles were evident in IFN-γ-treated astrocytes by 24 h after invasion. Overall, these data suggest that IFN-γ-activated astrocytes inhibitT. gondii by an as-yet-unknown mechanism.