ABSTRACTWe recently showed that the pyridinylimidazoles SB203580 and SB202190, drugs designed to block human p38 mitogen-activated protein kinase (MAPK) activation, also inhibited replication of the medically important intracellular parasiteToxoplasma gondiiin cultured human fibroblasts through a direct effect on the parasite. We now show that additional pyridinylimidazole and imidazopyrimidine p38 MAPK inhibitors inhibit intracellularT. gondiireplication in vitro and protect mice against fatalT. gondiiinfection. Mice surviving infection following treatment with p38 MAPK inhibitors were resistant to subsequentT. gondiichallenge, demonstrating induction of protective immunity. Thus, drugs originally developed to block human p38 MAPK activation are useful for treatingT. gondiiinfection without inducing significant immunosuppression. MAPK inhibitors combined with either of the approved anti-Toxoplasmadrugs sulfadiazine and pyrimethamine resulted in improved survival among mice challenged with a fatalT. gondiiinoculum. A MAPK inhibitor also treated mice infected with theMicrosporidiumparasiteEncephalitozoon cuniculi, suggesting that MAPK inhibitors represent a novel class of agents that may have a broad spectrum of antiparasitic activity. Preliminary studies implicate aT. gondiiMAPK homologue as the target of drug action, suggesting possibilities for more-selective agents.