Intracellular, inducible β-glucosidase from the cellulolytic fungus Sporotrichum (Chrysosporium) thermophile (ATCC 42464) was fractionated by gel chromatography or isoelectric focusing into components A and B. Enzyme A (molecular weight 440,000) had only aryl-β-glucosidase activity, whereas enzyme B (molecular weight 40,000) hydrolyzed several β-glucosides but had only low activity against o-nitrophenyl-β-d-glucopyranoside (ONPG). Both enzymes had temperature optima of about 50°C. The pH optimum was 5.6 for enzyme A and 6.3 for enzyme B, respectively. The Km (ONPG) value for enzyme A was 0.5 mM, and the corresponding values for enzyme B were 0.18 mM (ONPG) and 0.28 mM (cellobiose). Enzyme B, when tested with ONPG, showed substrate inhibition at a substrate concentration above 0.4 mM which could be released by cellobiitol and other alditols. Enzyme A was isoelectric at pH 4.48, and enzyme B was isoelectric at pH 4.64. Several inhibitors were tested for their action on the activity of enzymes A and B. Both enzymes were found to be concomitantly induced in cultures with either cellobiose or cellulose as carbon source.