Two chitinases, C-1 and C-2, were purified from the culture supernatant of Streptomyces griseus HUT 6037 by ammonium sulfate precipitation, Butyl-Toyopearl 650M chromatography, and chromatofocusing. Both enzymes had a molecular weight estimated to be 27,000 by SDS polyacrylamide gel electrophoresis, while their pIs were 7.7 and 7.3, respectively. The enzymes were active from pH 4.5 to 6.0 and their optimum temperature was 55 degrees C. They were stable between pH 6.5 and 10.0 and at temperatures below 50 degrees C. Chitinases C-1 and C-2 hydrolyzed chitin, colloidal chitin, glycol chitin, carboxymethyl chitin, 53% deacetylated chitosan, and (GlcNAc)(3-6), but did not hydrolyze 96% deacetylated chitosan and (GlcN)(n). The hydrolyzates of 53% deacetylated chitosan by these enzymes were separated by CM-Sephadex C-25 column chromatography. The products were analyzed structurally to elucidate the specificity of the chitinases. The oligosaccharides isolated had GlcNAc as the nonreducing end and GlcNAc or GlcN as the reducing end residues. These results indicate that the enzymes cleave both the N-acetyl-beta-D-glucosaminidic and the beta-glucosaminidic linkages in partially N-acetglated chitosan molecules.