Bacillus stearothermophilus IAM11001 produced three beta-galactosidases, beta-galactosidase I, II, and III (beta-gal I, II, and III), which are detectable by polyacrylamide (nondenatured) gel electrophoresis. By connecting restriction fragments of the chromosomal DNA to plasmid vectors, followed by transformation of Escherichia coli, two beta-galactosidase genes (bgaA and bgaB) located close to each other on the chromosome were isolated. Identification of the gene products and Southern hybridization analyses with a 2.7-kilobase-pair EcoRI fragment containing the bgaA gene as probe revealed that a single bgaA gene exists on the genome and that beta-gal II and beta-gal III consist of a common subunit (the bgaA gene product; molecular weight, 120,000), but differ in their assembly (beta-gal II is a dimer, and beta-gal III is a tetramer). The bgaB gene product (molecular weight, 70,000) in Bacillus subtilis harboring pHG5 (a hybrid plasmid consisting of pUB110 and a 2.9-kilobase-pair EcoRI fragment) was estimated to be the beta-gal I protein from its heat stability. Southern hybridization and immunological testing indicated that the two genes have no homology.