Work from several laboratories suggests a correlation between expression of cysteine proteinase activity and the cytopathic effect of virulent HM1 strain Entamoeba histolytica trophozoites on cultured cell monolayers. Consistent with this relationship, we find that L-6 trophozoites, mutants cloned from the HM1 parent strain, are deficient in both proteinase expression and cytopathic effect. Three other clones, with proteinase expression equal to or greater than that of the HM1 strain, express the cytopathic effect. Furthermore, a nontoxic specific proteinase inhibitor, Z-phenylalanyl-alanyl-CH2F, inhibits the cytopathic effect of live trophozoites in a dose-dependent manner. These results support the hypothesis that expression and release of the cysteine proteinase is an important factor in producing the cytopathic effect, presumably by its degradation of cell anchoring proteins.