The cytopathic effect (CPE) of southern African, British, and an Asian strain of Acanthamoeba was assessed using a system developed around 2 different mammalian cell (MC) lines. The time taken by the amebae to destroy cell cultures completely was shown to be dependent largely on the size of the amebic inoculum and the cell type. This highlights the need to assess carefully the behavior of cell lines prior to using them for cytopathic testing. Assays performed with conditioned medium collected from both MCs and amebic cells indicated that mechanical destruction may have been primarily responsible for the CPE. Furthermore, not all strains of Acanthamoeba lose cytopathogenicity after being passaged in axenic culture for extended periods. The use of MC cultures was shown to be an accurate, rapid, and repeatable means of assaying the CPE of strains of Acanthamoeba.