A new method for determining the specificity of hydrolysis of the linear binary heteropolysaccharide chitosan composed of (1-->4)-linked 2-acetamido-2-deoxy-beta-D-glucopyranose (GlcNAc; A-unit) and 2-amino-2-deoxy-beta-D-glucopyranose (GlcN; D-unit) residues is described. The method is based on the assignments of the 13C chemical shifts of the identity (A- or D-units) of the new reducing and non-reducing ends and the variation in their nearest neighbours, using low molecular weight chitosans with known random distribution of A- and D-units as substrate. A highly N-acetylated chitosan with fraction of acetylated units (FA) of 0.68 and a number-average degree of polymerization (DPn) of 30 was hydrolysed with hen egg-white lysozyme, showing that both the new reducing and non-reducing ends consisted exclusively of A-units, indicating a high specificity for A-units in subsites DL and EL on lysozyme. Our data suggests that the preceding unit of the reducing A-units, is invariable, and based on earlier studies, most probably an A-unit, while the unit following the non-reducing A-units can be either an A- or a D-unit. A more detailed study of the specificity of lysozyme at subsite DL was performed by hydrolyzing a more deacetylated chitosan (FA = 0.35 and DPn of 20) to a DPn of 9, showing that even for this chitosan more than 90% of the new reducing ends were acetylated units. Thus, lysozyme depolymerizes partially N-acetylated chitosans by preferentially hydrolyzing sequences of acetylated units bound to site CL, DL and EL of the active cleft, while there is no specificity between acetylated and deacetylated units to site FL. In addition, a moderately N-acetylated chitosan with fraction of acetylated units (FA) of 0.35 and a DPn of 20 was hydrolysed with Bacillus sp. No. 7-M chitosanase, showing that both the new reducing and non-reducing ends consisted exclusively of D-units. Our data suggests that the nearest neigbour to the D-unit at the reducing end is invariable, and based on earlier studies, most probably a D-unit, while the unit following the non-reducing D-units can be either an A- or a D-unit. We conclude that the Bacillus chitosanase hydrolyzes partially N-acetylated chitosan by preferentially attacking sequences of three consecutive deacetylated units, hypothetical subsites CC, DC and EC, where the cleavage occur between sugar units bound to subsites DC and EC. A hypothetical subsite FC on the chitosanase show no specificity with respect to A- and D-units. The new NMR method described herein offers a time and labour-saving alternative to the procedure of extensive hydrolysis of the binary heteropolysaccharide chitosan and subsequent isolation and characterization of the oligosaccharides.