Isoenzyme electrophoresis of three different enzyme systems was used to compare 71 strains assigned to the 15 currently recognized species of Acanthamoeba. A phylogenetic (cladistic) analysis of the zymograms indicated an arrangement of strains in 15 distinguishable lineages, but not all corresponding to current taxonomic assignments. Five of the groups corresponded to the recognized species A. castellanii, A. culbertsoni, A. griffini, A. lenticulata and A. royreba. But none of these groups consisted of only strains which had been previously assigned to each respective species. The type-equivalent strains for two species, A. hatchetti and A. tubiashi, were not closely aligned to any other strain and thus are considered to be monotypic. Strains of A. triangularis, A. astronyxis and A. palestinensis occurred together in a single group suggesting possible synonymy; however, on morphologic criteria, the strains assigned to these species are readily distinguishable. Strains assigned to A. polyphaga and A. rhysodes were interspersed throughout the other species groups. The strains of these two species were either misidentified or the species could not be recognized. Two groups previously not recognized as unique formed distinctive clusters which could be considered as new species. The analysis also made it possible to place strains which had previously been identified only to genus into species complexes. These results therefore suggest that previous criteria which have been used to classify Acanthamoeba are not adequate for fully resolving taxa at the species level.