Two putative alpha-galactosidase genes from rice (Oryza sativa L. var. Nipponbare) belonging to glycoside hydrolase family 27 were cloned and expressed in Escherichia coli. These enzymes showed alpha-galactosidase activity and were purified by Ni Sepharose column chromatography. Two purified recombinant alpha-galactosidases (alpha-galactosidase II and III; alpha-Gal II and III) showed a single protein band on SDS-PAGE with molecular mass of 42 kDa. These two enzymes cleaved not only alpha-D-galactosyl residues from the non-reducing end of substrates such as melibiose, raffinose, and stachyose, but also liberated the galactosyl residues attached to the O-6 position of the mannosyl residue at the reducing-ends of mannobiose and mannotriose. In addition, these enzymes clipped the galactosyl residues attached to the inner-mannosyl residues of mannopentaose. Thus, alpha-Gal II catalyzes efficient degalactosylation of galactomannans, such as guar gum and locust bean gum.