Enzymes with a-galactosidase activity are produced by many organisms, often in multiple forms. Here we compare the biochemical and hydrolytic properties of four major alpha -galactosidase forms (alpha -gal I-IV) that were purified from the culture filtrate of Aspergillus niger. alpha -Gal H, III and IV appear to be isoforms of the same enzyme, and N-terminal amino acid sequence data suggest that they are closely related or identical to A. niger AglB in family 27 of the glycosyl hydrolases. alpha -Gal I is a completely different enzyme that belongs to family 36. alpha -Gal I had an isoelectric point of 4.15 and appears to be a tetramer composed of four 94-kDa subunits. alpha -Gal H, Ed and IV were dimers with monomeric molecular masses of 64 kDa and isoelectric points of 4.5, 4.7 and 4.8, respectively. alpha -Gal H-IV were stable when incubated for 17 h at 50 degreesC and pH 2-5, whereas alpha -gal I was most stable at pH 5-6. All enzymes had maximal catalytic activity at pH 4.5 and 60 degreesC, and hydrolyzed melibiose, raffinose and stachyose. alpha -Gal H-IV also degraded galactomanno-oligosaccharides and released 66% of the galactose side groups from polymeric locust bean gum galactomannan. alpha -Gal I released galactose from locust bean gum only in combination with A. niger beta -mannosidase. Kinetic experiments showed that alpha -gal I hydrolyzed p-nitrophenyl-alpha -D-galactopyranoside and melibiose more efficiently than alpha -gal II-IV. The distinct hydrolytic and biochemical properties of alpha -gal I and alpha -gal II-IV further signifies the difference between alpha -galactosidases of family 27 and 36.