A recombinant beta-galactosidase from Caldicellulosiruptor saccharolyticus was purified with a specific activity of 211 U mg(-1) by using heat treatment and His-trap affinity chromatography. The native enzyme was an 80-kDa trimer with a molecular mass of 240 kDa. Maximum activity was observed at pH 6.0 and 80A(0)C, and the half-life at 70A(0)C was 48 h. The enzyme exhibited hydrolytic activity for p-nitrophenyl-beta-d-galactopyranoside (pNPGal), oNPGal, or lactose, whereas no activity for p-nitrophenyl-beta-d-glucopyranoside (pNPGlu), oNPGlu, or cellobiose. The catalytic residues E150 and E311 of beta-galactosidase from C. saccharolyticus were completely conserved in all aligned glycoside hydrolase family 42 beta-galactosidases. The results indicated that the enzyme was a beta-galactosidase. Galactose uncompetitively inhibited the enzyme. Glucose inhibition of the enzyme was the lowest among beta-galactosidases. When 50 g l(-1) galactose was added, the enzyme activity for pNPGal was reduced to 26%. When 400 g l(-1) glucose instead of galactose was added, the activity was reduced to 82%. When adding galactose (200 g l(-1)), only 14% of the lactose was hydrolyzed after 180 min. In contrast, the addition of glucose (400 g l(-1)) did not affect lactose hydrolysis, and more than 99% of the lactose was hydrolyzed after 120 min.