An intracellular beta-1,6-glucosidase (beta-D-glucoside glucohydrolase, EC 3.2.1.21) was produced semiconstitutively by Flavobacterium M64. This enzyme was purified 180-fold by fractionation with ammonium sulfate followed by chromatographies on carboxymethylcellulose, hydroxyapatite and Sephadex G-100. The final preparation appeared homogeneous on disc electrophoresis on polyacrylamide gel. The molecular weight of the enzyme was determined to be ca. 59 000 by Sephadex G-100 gel filtration and sodium dodecylsulfate-polyacrylamide gel electrophoresis. The optimum pH of the enzyme was 5.8 and the optimum temperature was 40 degrees C. The enzyme readily hydrolyzed oligomers with beta-a,6-glucosidic linkages, converting them to glucose. The Km values for gentio-biose, -triose, -tetraose and -pentaose were 2.8, 3.0, 4.2 and 4.6 times 10- minus 4 M, respectively. The rates of their hydrolyses decreased with increase in their chain lengths. The enzyme was concluded to be a beta-1,6-glucosidase from its substrate specificity, production of glucose, transferring ability and inhibition by glucono-delta-lactone. The enzyme activity was inhibited by Hg-2+, Cu-2+, Ag-+, Fe-3+, p-chloromercuribenzoate, N-ethylmaleimide, glucose and trishydroxyaminomethane (Tris) but not by ethylenediaminetetraacetic acid.