A -galactosidase gene (-Gal II) from Bifidobacterium adolescentis DSM 20083 was cloned into a pbluescript SK (–) vector and expressed in Escherichia coli. The recombinant enzyme was purified from the cell extract by anion-exchange and size-exclusion chromatography. -Gal II had a native molecular mass of 235 kDa and the subunits had a molecular mass of 81 kDa, indicating that -Gal II occurs as a trimer. The enzyme was classified as belonging to glycosyl hydrolase family 42. The optimal pH was 6.0 and the optimal temperature was 50°C, using p-nitrophenyl--d-galactopyranoside as a substrate. The K_m and V_max for Gal(1–4)Gal were 60 mM and 1,129 U/mg, respectively. The recombinant -Gal II was highly active towards Gal(1–4)Gal and Gal(1–4)Gal-containing oligosaccharides; only low activity was observed towards Gal(1–3)Gal, lactose, and Gal(1–3)GalOMe. No activity was found towards Gal(1–6)Gal, Gal(1–4)Man, Gal(1–4)Gal, Gal(1–3)Gal(1–4)Gal, cellobiose, maltose and sucrose. -Gal II was inhibited at high substrate concentrations (100 mg/ml) and no transglycosylation activity was found. At lower substrate concentrations (10 mg/ml) only low transglycosylation activity was found; the Gal/[Gal(1–4)]₂Gal peak area ratio was 9:1.