The nucleotide sequence was determined for the alkalophilic Bacillus sp. strain AM-001 beta-mannanase gene which produced two beta-mannanases (A and B) in Escherichia coli transformants. The putative beta-mannanase gene was 1,539 base pairs long and encoded a mature beta-mannanase protein of 487 amino acids and a signal peptide of 26 amino acids. The COOH-terminal amino acid of beta-mannanase A is an arginine residue located at amino acid 513 of the deduced amino acid sequence, and that of beta-mannanase B is a valine residue located at amino acid 365. Deletion derivatives having 1,098 base pairs from the ATG start codon maintained the beta-mannanase activity of the encoded polypeptide. However, clones harboring DNA fragments (1,051 base pairs) shorter than the gene which encoded beta-mannanase B (1,095 base pairs) did not exhibit the beta-mannanase activity. The simultaneous production of both beta-mannanases A and B in an E. coli transformant was demonstrated by the maxicell procedure.