We have synthesized beta-D-GlcNAc-(1-->4)-beta-D-GlcNAc-(1-->4)-beta-D-GlcNAc-(1-->4)-D-GlcN (2) through a partial N-acetylation reaction of chitosan tetramer 1 by a chitin deacetylase from Colletotrichum lindemuthianum ATCC 56676. The compound was purified from the mixture of acetylation products of 1 using cation-exchange column chromatography and amine-adsorption column chromatography, and its structure was estimated by 1H NMR and FABMS analyses. The enzymatic reaction allows a regioselectivity that is hard to achieve by chemical N-acetylation.