A systematic study of small free-living amoebae has been made under standardized and reproducible cultural conditions, and their pathogenicity has been tested in mice. Naegleria aerobia, Hartmannella culbertsoni and H. rhysodes are pathogenic; H. castellanii, H. astronyxis, H. palestinensis, H. glebae, H. exundans, H. vermiformis, Schizopyrenus russelli, Didascalus thorntoni and Tetramitus rostratus are non-pathogenic. Strains of H. culbertsoni and H. rhysodes are present in Indian soils. A classification of the order Amocbida Kent into families Schizopyrenidae Singh, Hartmannellidae Volkonsky, 1931 emend. Singh, 1952, and Endamoebidae (Calkins), based on nuclear division, is proposed, and the relation of this classification to previously defined families and genera of amoebae and its bearing on phylogeny are discussed. Metronidazole and other anti-amoebic drugs are ineffective against N. aerobia and H. culbertsoni in vitro and in meningo-encephalitis in mice.