Although beta-D-fucosidase (beta-D-fucoside fucohydrolase, EC 3.2.1.38) has been isolated from various sources, the identity of this enzyme is still not settled. We have purified a specific beta-D-fucosidase in electrophoretically homogeneous form crude extracts of Aspergillus phoenicis by polyethyleneglycol 6000-phosphate buffer aqueous two-phase separation, and successive chromatography on DEAE-Sephadex A-50, hydroxyapatite and Sephadex G-100 columns. The molecular weight of the enzyme was estimated to be 57000 by SDS-polyacrylamide gel electrophoresis and 50000 to 60000 by gel filtration on Sephadex G-100. The enzyme showed optimum coside were 2.4mmol/L, and 1.28 mumol min-1 the pH range 5.5-6.5 and below 35 degrees C. The Km and the Vmax values for pNP-beta-D-fucoside were 2.4mmol/L, and 1.28 mumol.min-1.mg-1 respectively. The enzyme was strongly inhibited by sulfhydryl group reagents, PCMB-NEM and iodoacetate. It was also inhibited by EDC, DEP and NBS. Thus, -SH, -COOH groups, histidyl and tryptophyl residues were essential for enzyme activity. The purified beta-D-fucosidase showed high specificity toward p-nitrophenyl beta-D-fucoside. The enzyme was inhibited by D-fucose and D-fucono-gamma-lactone, but not by D-galactose, D-galactono-gamma-lactone, D-glucose or D-glucono-gamma-lactone; the latter compounds are specific inhibitors of beta-D-galactosidase and beta-D-glucosidase respectively. Thus, this enzyme is the most strictly specific beta-D-fucosidase when compared with those previously reported.