Glucosidase II (G-II) is a glycoprotein-processing enzyme that successively cleaves two alpha1,3-linked glucose residues from N-linked oligosaccharides in the endoplasmic reticulum. G-II is a heterodimer whose alpha-subunit contains a glycosidase active site, but the function(s) of the beta-subunit remain poorly defined. We report here an in vivo enzymatic analysis using gene disruptants lacking either the G-II alpha- or beta-subunit in the filamentous fungus Aspergillus oryzae. Using synthetic oligosaccharides as probes, G-II activity of the membranous fraction of the gene disruptants was investigated. The fraction lacking the beta-subunit retained hydrolytic activity toward p-nitrophenyl alpha-D-glucopyranoside but was inactive toward both Glc(2)Man(9)GlcNAc(2) and Glc(1)Man(9)GlcNAc(2). When the fraction containing the beta-subunit was added to the one including the alpha-subunit, the glucosidase activity was restored. These results suggested that the beta-subunit confers the substrate specificity toward di- and monoglucosylated glycans on the glucose-trimming activity of the alpha-subunit.