Acanthamoeba spp. are free-living amebae associated with amebic keratitis and chronic granulomatous amebic encephalitis. The present studies were undertaken to compare the pathogenicity of three species of Acanthamoeba in B6C3F1 mice after intranasal challenge with Acanthamoeba-induced cytopathogenicity for different macrophage populations. The ability of murine macrophage cell lines and activated murine peritoneal macrophages to lyse Acanthamoeba has been assessed by coincubating macrophages with 3H-uridine labeled amebae. Conversely, destruction of macrophages by Acanthamoeba was determined by measuring the release of chromium-51 from radiolabeled macrophages. Acanthamoeba culbertsoni, which is highly pathogenic for mice, destroys macrophage cultures in vitro. Activated primary peritoneal macrophages were more resistant to Acanthamoeba-mediated destruction than macrophage cell lines activated in vitro. Activated macrophages were capable of limited destruction of Acanthamoeba polyphaga and Acanthamoeba castellanii. Acanthamoeba-specific antibodies increased the amebicidal activity of activated macrophages. Macrophage-mediated destruction was by contact-dependent cytolysis and by ingestion of amebae. Conditioned medium obtained from macrophage cultures after treatment with lipopolysaccharide and interferon gamma was neither cytolytic nor cytostatic for Acanthamoeba spp. Purified recombinant cytokines including tumor necrosis factor alpha, interleukin 1 alpha, and interleukin 1 beta, alone or in combination, were not cytolytic for Acanthamoeba trophozoites.