A novel alpha-galactosidase, designated alpha-galactosidase II, was isolated from the culture filtrate of Mortierella vinacea. The molecular size of the purified enzyme estimated by gel filtration was 60 kDa, which agreed with that, 51-62 kDa, estimated by SDS-PAGE. The enzyme was thermolabile at neutral pH, but the addition of BSA to the enzyme solution at the concentration of 0.01% increased its stability considerably. The enzyme appears to be novel because it showed a distinct substrate specificity from other microbial alpha-galactosidases on galactomanno-oligosaccharides, prepared from galactomannan, that is, the enzyme liberated not only side-chain alpha-galactosyl residue from 6(3)-mono-alpha-D-galactopyranosyl-beta-1,4-D-mannotetraose but also terminal alpha-galactosyl residue from 6(3)-mono-alpha-D-galactopyranosyl-beta-1,4-D-mannotriose. In addition, the enzyme acted on galactomannans effectively. alpha-Galactosidase II cDNA was cloned and its nucleotides sequenced. The deduced amino acid sequence showed that the mature enzyme consisted of 376 amino acid residues with a molecular mass of 41,334 Da. The derived amino acid sequence of the enzyme showed 31-49% sequence similarity with those of alpha-galactosidases from other origins.