The substrate specificity of N-acetylhexosaminidase (E.C. 3.2.1.51) from Aspergillus oryzae was examined using p-nitrophenyl 6- O-sulfo- N-acetyl-β- d-glucosaminide (6- O-sulfo-GlcNAc- O- pNP) as the glycosyl donor and a series of β- d-glucopyranosides and N-acetyl-β- d-glucosaminides with variable aglycons at the anomeric positions as the acceptors. When β- d-glucopyranosides with methyl (CH 3), allyl (CH 2CH CH 2), and phenyl (C 6H 5) groups at the reducing end were used as the acceptors, this enzyme transferred the 6- O-sulfo-GlcNAc moiety in the donor to the location of O-4 in these glycosyl acceptors with a high regioselectivity, producing the corresponding 6- O-sulfo- N-acetylglucosaminyl β- d-glucopyranosides. However, β- d-glucopyranose lacking aglycon was a poor substrate for transglycosylation. This A. oryzae enzyme could also accept various N-acetyl-β- d-glucosaminides carrying hydroxyl (OH), methyl (CH 3), propyl (CH 2CH 2CH 3), allyl (CH 2CH CH 2) and p-nitrophenyl ( pNP; C 6H 4–NO 2) groups at their aglycons, yielding 6- O-sulfo- N-acetylglucosaminyl-β(1→4)-disaccharide products.