The substrate specificity of an extracellular beta-glucosidase (BGL) from cellulose-degrading culture of the white-rot fungus Phanerochaete chrysosporium was investigated, using a variety of compounds with beta-glucosidic linkages. Amino acid sequencing data for the purified BGL showed that the enzyme is identical to the glycoside hydrolase (GH) family 3 BGL of the same fungus previously reported [Li, B. and Renganathan, V., Appl. Environ. Microbiol., 64, 2748-2754 (1998)]. The BGL can hydrolyze both cellobiose and cellobionolactone, but cellobionolactone was hydrolyzed very much more slowly than cellobiose. Moreover, cellobionolactone inhibited cellobiose hydrolysis by the BGL, suggesting that this enzyme cannot cooperate with cellobiose dehydrogenase (CDH) in cellulose degradation by P. chrysosporium. In addition to cellobiose, BGL utilized various glucosyl-beta-glucosides, such as sophorose, laminaribiose and gentiobiose, as substrates. Among the four substrates, laminaribiose (beta-1,3-glucosidic linkage) was hydrolyzed most effectively. Moreover, the hydrolytic rate of laminarioligosaccharides increased proportionally to the degree of polymerization (DP), and the activity of BGL even towards laminarin with an average DP of 25 was similar to that towards laminaripentaose (DP 5). Therefore, we conclude that the extracellular BGL from P. chrysosporium is primarily a glucan 1,3-beta-glucosidase (EC 3.2.1.58), which might play a role on fungal cell wall metabolism, rather than a beta-glucosidase (EC 3.2.1.21), which might be involved in the hydrolysis of beta-1,4-glucosidic compounds during cellulose degradation.