Bacillus circulans WL-12, isolated as a yeast cell wall-lytic bacterium, secretes a variety of polysaccharidedegrading enzymes into culture medium. When chitinases of the bacterium were induced with chitin, six distinct chitinase molecules were detected in the culture supernatant. These chitinases (Al, A2, Bi, B2, C, and D) showed the following distinct sizes and isoelectric points: Mr 74,000, pl 4.7 (Al); Mr 69,000, pl 4.5 (A2); Mr 38,000, pl 6.6 (Bi); Mr 38,000, pl 5.9 (B2); Mr 39,000, pl 8.5 (C); and Mr 52,000, pl 5.2 (D). Among these chitinases, Al and A2 had the highest colloidal-chitin-hydrolyzing activities. Chitinase Al showed a strong affinity to insoluble substrate chitin. Purified chitinase Al released predominantly chitobiose [(GlcNAc)2] and a trace amount of N-acetylglucosamine (GlcNAc) from colloidal chitin. N-terminal amino acid sequence analysis of chitinases Al and A2 indicated that chitinase A2 was generated from chitinase Al, presumably by proteolytic removal of a C-terminal portion of chitinase Al. Since chitinase A2 did not have the ability to bind to chitin, the importance of the C-terminal region of chitinase Al to the strong affinity of chitinase Al to substrate chitin was suggested. Strong affinity of the chitinase seemed to be required for complete degradation of insoluble substrate chitin. From these results, it was concluded that chitinase Al is the key enzyme in the chitinase system of this bacterium.Chitin, an insoluble linear P-1,4-linked polymer of Nacetylglucosamine (GlcNAc), is one of the most abundant polysaccharides in nature. It is a common constituent of insect exoskeletons, shells of crustaceans, and fungal cell walls. These organisms containing constituent chitin produce chitinases (EC 3.2