We purified and characterized a thermophilic beta-galactosidase from Thermus sp. A4 isolated from the Atagawa hot spring (Shizuoka, Japan). The enzyme was monomeric, and its molecular mass was estimated to be 75 kDa by SDS-polyacrylamide gel electrophoresis. The enzyme was extremely thermostable and retained its full activity after incubation at 70 degrees C for 20 h. The Km observed were 5.9 mM for ortho-nitrophenyl beta-D-galactopyranoside and 19 mM for lactose. We cloned and analyzed the complete sequence of the gene encoding this enzyme. It was found to consist of 645 amino acid residues. We propose that this enzyme and seven other unclassified beta-galactosidases are new members of family 42 of the glycosyl hydrolases.