A strain, KA/S2, isolated from Korean soil and morphologically assigned to Acanthamoeba castellanii, was characterized by isoenzyme analysis, and total proteins profile, and mitochondrial (Mt) DNA restriction fragment length polymorphism (RFLP), and compared with four reference strains assigned to the species (the authenitic Castellani, Neff, Ma, and Chang strains). It was found that four isoenzyme, total proteins, and Mt DNA RFLP patterns by eight restriction endonucleases of the strain KA/S2 were identical with those of the Neff strain, isolated from soil of California, USA. The Chang strain was unique in its morphology and total protein patterns. Interstrain polymorphisms of isoenzyme profiles and Mt DNA RFLP patterns were observed among the Castellani, Neff, Ma, and Chang strains. Mt DNA RFLP was confirmed to be highly appropriate for the strain characterization and identification of Acanthamoeba spp.