A novel -glucosidase with an apparent subunit mass of 59 ± 0.5 kDa was purified from protein extracts of Rhizobium sp. strain USDA 4280, a nodulating strain of black locust (Robinia pseudoacacia L), and characterized. After purification to homogeneity (475-fold; yield, 18%) by ammonium sulfate precipitation, cation-exchange chromatography, hydrophobic chromatography, dye chromatography, and gel filtration, this enzyme had a pI of 4.75 ± 0.05. The enzyme activity was optimal at pH 6.0 to 6.5 and 35°C. The activity increased in the presence of NH₄⁺ and K⁺ ions but was inhibited by Cu²⁺, Ag⁺, Hg⁺, and Fe²⁺ ions and by various phenyl, phenol, and flavonoid derivatives. Native enzyme activity was revealed by native gel electrophoresis and isoelectrofocusing-polyacrylamide gel electrophoresis with fluorescence detection in which 4-methylumbelliferyl -glucoside was the fluorogenic substrate. The enzyme was more active with -glucosides substituted with aromatic aglycones than with oligosaccharides. This -glucosidase exhibited Michaelis-Menten kinetics with 4-methylumbelliferyl -D-glucopyranoside (K_m, 0.141 µM; V_max, 6.79 µmol min¹ mg¹) and with p-nitrophenyl -D-glucopyranoside (K_m, 0.037 µM; V_max, 2.92 µmol min¹ mg¹). Maltose, trehalose, and sucrose were also hydrolyzed by this enzyme.