Twenty mollicute strains isolated primarily from insect hosts were characterized and arranged into eight new species in the genus Mesoplasma. Morphological examination of the organisms by electron and dark-field microscopic techniques revealed that the cells of each strain were small, nonhelical, nonmotile, pleomorphic, and coccoid and that each cell was surrounded by a single cytoplasmic membrane with no evidence of a cell wall. Although the new mollicutes grew well in media containing horse or fetal bovine serum, growth in serum-free or cholesterol-free medium occurred only when the medium contained 0.04% polyoxyethylene sorbitan (Tween 80). The optimum temperature for growth was usually 30 degrees C, but multiplication generally occurred over a temperature range of 10 to 32 degrees C. All strains catabolized glucose. Most strains did not hydrolyze arginine or urea, although three related strains isolated from fireflies (the strain PUPA-2T [T = type strain] group) did hydrolyze arginine. The genome sizes ranged from 825 to 930 kbp, and the DNA base compositions (guanine-plus-cytosine contents) ranged from 26.5 to 31.6 mol%. The proposed type strains of the eight new species were not serologically related to the type strains of four other Mesoplasma species, five Entomoplasma species, 11 Acholeplasma species, and 100 Mycoplasma species and subspecies. Strain PS-1 (= ATCC 49582) is the type strain of Mesoplasma pleciae sp. nov., strain PUPA-2 (= ATCC 49581) is the type strain of Mesoplasma photuris sp. nov., strain YJS (= ATCC 51578) [corrected] is the type strain of Mesoplasma syrphidae sp. nov., strain CHPA-2 (= ATCC 49578) is the type strain of Mesoplasma chauliocola sp. nov., strain ELCA-2 (= ATCC 49579) is the type strain of Mesoplasma corruscae sp. nov., strain GRUA-1 (= ATCC 49580) is the type strain of Mesoplasma grammopterae sp. nov., strain BARC 779 (= ATCC 49583) is the type strain of Mesoplasma coleopterae sp. nov., and strain BARC 857 (= ATCC 49584) is the type strain of Mesoplasma tabanidae sp. nov.