Acanthamoeba keratitis is a sight-threatening complication of corneal trauma or contact lens wear. Although the majority of corneal isolates of Acanthamoeba belong to Group II in the Pussard-Pons classification based on cyst morphology, they have been placed in at least six species and their genetic relatedness is uncertain. The aim of this study was to determine the virulence of, and the relationship among, strains derived from the cornea, the nasal mucosa, and other environmental sources. To assess virulence, 10(4) trophozoites of each strain were incubated with monolayers of human corneal fibroblasts. By day 7, 12 of 29 strains tested had induced significant cytopathic changes. In addition, inocula of 10(4) cysts or trophozoites with 10(6) Corynebacterium xerosis were injected into the corneas of Porton rats; 11 amoebic strains induced infection within 7 days. The correlation between the virulence of trophozoites in vitro and in vivo was 86%. Using allozyme electrophoresis, 23 Acanthamoeba strains clustered into 5 major phylogenic divisions. Three divisions contained one or more strains that were virulent in the rat cornea. Virulent Pussard-Pons Group II strains clustered tightly to a fixed allelic difference of 13.6%. The eight corneal isolates clustered to 33%, dividing into three lineages. Five avirulent nasal isolates were strongly differentiated from other Group II strains. The results were not in accord with species designations based primarily on morphological criteria. These data suggest that particular subsets of Acanthamoeba strains are virulent in the human cornea.