An exo-1,4-beta-glucosidase (EC 3.2.1.74; G3ase) was obtained from the supernatant of cultured Acetobacter xylinum subsp. sucrofermentans BPR2001 and purified to homogeneity by ammonium sulfate precipitation, cation-exchange, gel-filtration and hydrophobic interaction chromatography. The enzyme migrated to a position corresponding to 81.2kDa on SDS-polyacrylamide gel electrophoresis under both non-reducing and reducing conditions, suggesting that this enzyme is a monomer polypeptide. The isoelectric point was 6.0. N-Bromosuccinimide inhibited the activity of exo-1,4-glucosidase completely, whereas sulfhydryl reagents did not. The K-m and V-max for the hydrolysis of cellotriose as substrate were 3.7 mM and 7.4 mu mol/min/mg, respectively. The enzyme specifically cleaved the non-reducing ends of beta-glucosyl linkages of cellotriose or larger cello-oligosaccharides, 4-methylumberiferryl- and p-nitrophenyl-beta-D-glucosides, but cellobiose was hydrolyzed only slightly and salicin not at all. The enzyme catalyzes the hydrolysis of glucosidic linkages in such a manner that the product retains the anomeric configuration of the substrate.