Streptomyces lividans 66 has three distinct chitinase genes. The nucleotide sequence of one gene for chitinase (chiB) from S. lividans was determined, and the structure and expression of the gene were analyzed. The structural gene consisted of 1,830 bp encoding 610 amino acid residues. Comparison of the deduced amino acid sequence of chitinase B with those of chitinases of other bacteria revealed a domain structure from N-terminal to C-terminal of the following order : signal peptide, substrate-binding domain, type III repeating unit, and catalytic domain. When the three chitinase genes from S. lividans (chiA, chiB, chiC) were compared with one another, the overall similarities between the nucleotide sequences of chiA and chiB and between the amino acid sequences deduced from them were found to be 49% and 59%, respectively, whereas chiC showed no relatedness to either chiA of chiB. It is, therefore, suggested that chiA and chiB diverged relatively recently, chiB of S. lividans was found to be more than 90% similar of chi01 of Streptomyces olivaceoviridis in both nucleotide and amino acid sequences, and chiC of S. lividans was found to be almost identical to chiA and S. plicatus. Southern hybridization studies conducted using chiA, chiB, and chiC of S. lividans as probes against genomic DNA from several Streptomyces strains revealed that these genes of S. lividans are distributed and highly conserved among the genus Streptomyces. Although a pair of direct repeat sequences similar to those found in the promoter region of other chitinase genes of Streptomyces are present in chiB, the motif is least conserved in chiB. The level of expression of chiB was shown to be lower than that of either chiA or chiC. It is assumed that chitinases A and C play a major role in chitin degradation in S. lividans.