Alpha-D-galactosidase gene (agal) of Bifidobacterium breve 203 was cloned into temperature expression vector pBV220 and transformed into E. coli. The recombinant plasmid pBVagal was induced to express with temperature. The specific activities of recombinant enzyme Agal in E. coli DH5alpha, E. coli DH10B and E. coli BL21 were 28.08 U/mg, 19.44 U/mg and 13.85 U/mg, respectively. The recombinant plasmid pBVagal is more stable in E. coli BL21. The molecular weight of Agal as determined by SDS-PAGE was about 67 kD. The optimum pH of Agal was pH 4.0-4.4, and it was stable between pH 3.6 and 6.0 (kept at 4 degrees C overnight). The optimum temperature of Agal was 45 degrees C, and it was stable below 40 degrees C (incubated for 30 min). Km-values for p-nitrophenyl-alpha-galactopyranoside (pNPGal) and melibiose were calculated with 1.43 mmol/L and 261 mmol/L, respectively. No transgalactosylation activity was found when Agal hydrolyzed melibiose or raffinose. The results suggest that Agal is much different from reported alpha-D-galactosidase from Bi. breve 203. Agal is another kind of alpha-D-galactosidase in the same bifidobacteria strain.