An intracellular β-1, 4-D-glucosidase (EC 3.2.1.21) was isolated from the mutant strain HP-3 ofStreptomyces lividans66 which produced about 12 times more enzyme than the wild-type strain. The purification was carried out by anion exchange column chromatography followed by high-performance liquid chromatography on DEAE and on molecular sieve columns. The enzyme is glycosylated and has an apparent Mrof 51 000 and a pI of 4.3. Its activity was optimal at pH 6.5 and at a temperature of 40 °C. TheKmand theVmaxon cellobiose were 3.1 mM and 65.6 μmol min−1mg−1of enzyme.Key words: β-glucosidase,Streptomyces lividans, purification, characterization.