N-Acetyl-β-D-glucosaminidase [EC 3. 2.1. 30] of Aspergillus oryzae was shown to have strong transglycosylation activity, and di- and trisaccharides were produced by the transfer reaction of this enzyme using a high substrate concentration. Using phenyl N-acetyl-β-D-glucosaminide as substrate, two disaccharides were obtained and their structures were determined by permethylation, periodate oxidation, and digestion with enzymes. The results showed that this enzyme transferred the N-acetylgluco-samine residue from the substrate to the hydroxyl group at carbon 4 or 6 of the substrate, the respective products being formed in a ratio of about 13: 1. When phenyl N-acetyl-β-D-galactosaminide was used as substrate, the transfer reaction hardly occurred. 3-O-Methylation or 6-O-methylation of the substrate, phenyl N-acetyl-β-D-gluco-saminide, had no effect on the transfer reaction using methanol as acceptor. The reaction was only slightly influenced by the pH of the reaction medium.