In a previous study, we found a unique hydrocellulase from the culture filtrate of Aspergillus aculeatus No. F-50, with insoluble cellooligosaccharide used as substrate of the enzyme. On the other hand, the reaction products from cellulosic substances by the crude enzyme of this strain were almost exclusively monosaccharides. This result might be attributed to the activity of β-glucosidase, and in this connection, the β-glucosidase of A. aculeatus was studied. This paper describes the purification and unique properties of the β-glucosidases from this fungus. β-Glucosidase activity was assayed by incubation with 0.5% salicin as substrate in 0.05 M sodium acetate buffer, pH 5.0, at 37°C for 10 min. The reducing sugars were determined by the Somogyi-Nelson method with glucose as standard. One unit of enzyme activity was defined as the amount of enzyme which released 1μmol of reducing sugar per min. The crude enzyme was prepared as described previously and loaded on a column (5×51 cm) of DEAE-Sephadex A-50 equilibrated with 0.03 M sodium phosphate buffer, pH 6.0, followed by gradient elution with decreasing pH from 6.0 to 3.5, yielding three β-glucosidase peaks named β-glucosidase-1, -2 and -3. The three enzymes were further purified by SP-Sephadex C-50, Bio Gel P-150 column chromatography and isoelectric focusing, and appeared homogeneous on polyacrylamide gel disc electrophoresis. The isoelectric points of β-glucosidase-1, -2 and -3 were pH 4.7, 4.3 and 3.6, respectively. Substrate specificities showed that β-glucosidase-3 was highly active on salicin but less active on cellobiose and only slightly active on insoluble cellooligosaccharide. On the contrary, both β-glucosidase-1 and -2 were potently active not only on salicin, but also on cellobiose and even insoluble cellooligosaccharide, but less active on CM-cellulose or swollen cellulose. The reaction product from insoluble cellooligosaccharide by both enzymes was only glucose. Consequently, β-glucosidase-3 closely resembles β-glucosidase from Trichoderma viride with respect to substrate specificity. β-Glucosidase-1 and -2 seemed to be similar to exoglucanase obtained from T. viride but differed in hydrolysis of CM-cellulose, as they were almost unable to hydrolyze CM-cellulose. Therefore, both enzymes of A. aculeatus should be classified as a new type of exo-β-glucosidase. β-Glucosidase-1 and -2 appear to play an important role in producing glucose from cellulose. More broad and detailed investigation on the three β-glucosidases will be reported on elsewhere.