In the central nervous system, cytokine-activated microglia play a crucial role in host defence against Toxoplasma gondii infections. In this study, the effect of recombinant tumor necrosis factor (rTNF)-alpha and prolactin (PRL) on T. gondii infection in microglia was examined. Pretreatment of microglia with rTNF-alpha and PRL induced toxoplasmastatic activity, the intracellular killing of T. gondii and the release of interleukin (IL)-1 beta IL-3 and IL-6: 50% of the intracellular killing was abrogated by anti-ICAM-1 monoclonal antibodies, whereas more than 54 or 87% of toxoplasmastatic activity was reversed by anti-IL-3 or IL-6 monoclonal antibodies. In addition, the treatment of microglia with either rIL-3 or rIL-6, in the absence or presence of rTNF-alpha significantly limited T. gondii replication. Inasmuch as either NMA or S-M-ITU affected cytokine-activated toxoplasmastatic activity during the infection phase, the NO-dependent pathway itself appears not to be directly involved in the parasitostatic activity. These findings suggest that TNF-alpha and PRL up-regulate the expression of ICAM-1 and the production of endogenous IL-6 and IL-3 by microglia, which could induce anti-parasitic functions against T. gondii infection in the brain.