The recombinant catalytic alpha-subunit of N-glycan processing glucosidase II from Schizosaccharomyces pombe (SpGII alpha) was produced in Escherichia coli. The recombinant SpGII alpha exhibited quite low stability, with a reduction in activity to <40% after 2-days preservation at 4 degrees C, but the presence of 10% (v/v) glycerol prevented this loss of activity. SpGII alpha, a member of the glycoside hydrolase family 31 (GH31), displayed the typical substrate specificity of GH31 alpha-glucosidases. The enzyme hydrolyzed not only alpha-(1 -> 3)-but also alpha-(1 -> 2)-, alpha-(1 -> 4)-, and alpha-(1 -> 6)-glucosidic linkages, and p-nitrophenyl alpha-glucoside. SpGII alpha displayed most catalytic properties of glucosidase II. Hydrolytic activity of the terminal alpha-glucosidic residue of Glc(2)Man(3)-Dansyl was faster than that of Glc(1)Man(3)-Dansyl. This catalytic alpha-subunit also removed terminal glucose residues from native N-glycans (Glc(2)Man(9)GlcNAc(2) and Glc(1)Man(9)GlcNAc(2)) although the activity was low.