Comparative studies were conducted on the structure, nutrition, protein composition, immunology, and effect on cell cultures of Acanthamoeba sp. (Lilly A-1 strain), A. castellanii (Singh and Neff strains), A. astronyxis, A. comandoni, A. polyphaga, A. terricola, Hartmannella vermiformis, and Naegleria gruberi. Lilly A-1 strain of Acanthamoeba received special attention owing to its pathogenicity for experimental animals. Distinct differences were noted in structure, nutrition, and antigenic composition of Acanthamoeba spp. and Hartmannella, and it was concluded that their recognition as separate genera is justified. With the exception of A. terricola, all species of Acanthamoeba could be differentiated by cyst structure. Cysts of A. terricola closely resembled those of A. castellanii Singh strain, and close antigenic relationships between these 2 species were demonstrated by gel diffusion and immunoelectrophoresis (IEP); it was concluded that the 2 amebae belong in the same species. The pathogenic Acanthamoeba sp, Lilly strain differed from the nonpathogenic A. castellanii Singh strain (a) cyst structure; (b) protein distribution patterns (on disc electrophoresis);(c)soluble and particulate antigens (on gel diffusion, IEP, complement fixation, and immobilization tests); (d) capacity to induce cell-free plaques and other cytopathic effect (CPE) in mammalian monlayer cell cultures; (e) elimination of a phospholipase, responsible for some of the CPE, into the culture medium. Acanthamoeba sp. Lilly strain, which liberated more phospholipase, produced more CPE. Acanthamoeba sp. Lilly strain differed also from other species of this genus in cyst structure and antigenic composition. It was concluded, therefore, that, following the recommendation of Singh & Das, it ought to be placed in a separate species, Acanthamoeba culbertsoni.