Glycosidases are valuable catalysts for the synthesis of a wide array of di- and oligosaccharides. Herein we report on the use of β-1-N-acetamido-2- acetamido-2-deoxy-d-glucopyranose, the simple model of the N-glycoprotein linkage region, as a novel acceptor for disaccharide synthesis catalyzed by β-N-acetylhexosaminidases from Aspergillus oryzae and mung beans (Vigna radiata) under transglycosylation as well as reversed hydrolysis. The exclusive formation of the disaccharide model, β-d-GlcNAc-(1→4)-d- GlcNAcβNHAc, by transglycosylation and the corresponding (1→6) analog under reversed hydrolysis both in reasonable yields demonstrates the versatility of β-N-acetylhexosaminidase from Aspergillus oryzae. The efficacy of the enzyme from Vigna radiata for synthesis has been demonstrated for the first time. This is also the first report on the use of derivatized sugars as co-reactants in glycosidase catalyzed reversed hydrolysis mode of synthesis. The excellent (1→6) selectivity of mung beans β-N-acetylhexosaminidase under transglycosylation and that from Aspergillus oryzae under reversed hydrolysis would prove to be very useful. © 2004 Elsevier Ltd. All rights reserved.