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Summary: Strains of three groups of Mycoplasma with different nutritional requirements were analysed for β-glucosidase activity. The enzyme was found in the cell membrane of Mycoplasma laidlawii strain b and M. gallisepticum strain J, which metabolize carbohydrates; negligible β-glucosidase activity was found in M. hominis strain 07 which does not metabolize carbohydrates. βglucosidase in all strains was inactivated by heating at 56° for 30 min. or at 100° for 10 min. With M. laidlawii strain B as the test organism, βglucosidase was found to possess the following characteristics: pH optimum, 6·8; optimum temperature, 30°; no dialysable cofactors required; phosphate ions had no effect on activity; enzyme was specific for substrates (glucosides) with β configuration; aglycon of the glucoside must be an aryl group; enzyme exhibited absolute specificity for carbon atoms number 4 and 5 of the glycon. The presence of β-glucosidase in M. laidlawii strain B and M. gallisepticum strain J is assumed to be involved in the synthesis and hydrolysis of carotenyl glucoside and sterol glucoside by these organisms.