Strain PYAN-1T (T = type strain), which was isolated from a pupal gut of the firefly beetle Pyractonema angulata, and strains PIMN-1T and PIPN-2T, which were isolated from guts of adult Photinus marginalis and Photinus pyralis fireflies, respectively, were demonstrated to be sterol-requiring mollicutes. Cells of the three strains were shown by electron and dark-field microscopy to be small, pleomorphic, nonhelical, nonmotile bodies surrounded by single membranes. No evidence of a cell wall was observed, and the organisms were not susceptible to 500 U of penicillin per ml. The three strains grew rapidly in SP-4 broth medium. Strains PIMN-1T and PIPN-2T grew in medium supplemented with bovine serum fraction, but strain PYAN-1T did not. All three strains grew on solid media when the cultures were incubated aerobically, but only strains PYAN-1T and PIPN-2T formed colonies when anaerobic conditions were employed. The three strains catabolized glucose but hydrolyzed neither arginine nor urea. All of the strains grew at temperatures of 18 to 32 degrees C; strains PYAN-1T and PIMN-1T also grew at 10 degrees C. The optimal temperature for growth for strains PYAN-1T and PIPN-2T was 30 degrees C; strain PIMN-1T grew equally well at 30 or 32 degrees C. None of the three strains grew at 37 degrees C. The genome sizes of strains PYAN-1T, PIMN-1T, and PIPN-2T were about 527 (478 to 589), 570 (480 to 630), and 762 (635 to 871) megadaltons, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)