The hydrolytic specificities of chitosanases were determined using N¹,N⁴-diacetylchitohexaose [(GlcN)₂-GlcNAc-(GlcN)₂-GlcNAc]. The results for the hydrolytic specificities of chitosanases belonging to subclasses I, II, and III toward chitohexaose and N¹,N⁴-diacetylchitohexaose agreed with previous results obtained by analysis of the hydrolysis products of partially N-acetylated chitosan. N¹,N⁴-Diacetylchitohexaose is a useful substrate to determine the hydrolytic specificity of chitosanase. On the other hand, chitosanases from Amycolatopsis sp. CsO-2 and Pseudomonas sp. A-01 showed broad cleavage specificity. They cleaved both the GlcNAc-GlcN and the GlcN-GlcNAc bonds in addition to the GlcN-GlcN bond in the substrate. Thus, both enzymes were new chitosanases. The chitosanases were divided into four subclasses according to their specificity for hydrolysis of the β-glycosidic linkages in partially N-acetylated chitosan.