β-Xylosidase was purified 662 fold from a culture filtrate by ammoniumsulfate fractionation, gel filtration on Biogel P-100, DEAE-Sephadexchromatography, and gel filtration on Sephadex G- 200. With isoelectric focusing, the purified β-xylosidase found to be homogeneous on SDS (sodium dodecyl sulfate) polyacrylamide gel electrophoresis. The molecular weight was estimated by gel filtration to be 240, 000, and 1 16, 000 by SDS polyacrylamide gel electrophoresis. The purified fixylosidase had an isoelectric point at pH 3.25, and contained 4% carbohydrate residue. The optimum pH was found to be in the range of 4.5-5, and the optimum temperature was 55°C. The enzyme activity was inhibited by Hg2+, SDS, and 7V-bromosuccinimide at a concentration of 1×10-3 m, andalso by/7-chloromercuribenzoate at a concentration of 1×10-4 m. The purified enzyme hydrolyzed phenyl β-D-x loside (ko = 302.6 sec-1), p-nitrophenyl β-D-xyloside (ko =438.9 sec-1), onitrophenyl β-D-xyloside (Ko=431.0 sec-1), p-chlorophenyl β-D-xyloside (KO=207.9 sec-1), ochlorophenyl β-D-xyloside (KO=211.8 sec-1), p-methylphenyl β-D-xyloside (KO=96.5 sec-1), omethylphenyl β-D-xyloside (KO=83. 1 sec-1), p-methoxyphenyl β-D-xyloside (KO =99.3 sec-1), omethoxyphenyl β-D-xyloside (KO= 100.0 sec-1), xylobiose (ko=992A sec-1), xylotriose (Ko= 1321.9 sec-1), xylotetraose (KO=789.7 sec-1) and xylopentaose (KO=508.0 sec-1). On enzymic hydrolysis ofphenyl β-D-xyloside, the reaction product was found to be β-D-xylose with retention of the configuration. The purified β-xylosidase was practically free of α-xylosidase and β-glucosidase activities.