The substrate specificities of three Penicillium simplicissimum ol-galactosidases, AGLI, AGLII, and AGLIII, were determined by using various isolated galactose-containing oligosaccharides and polymeric galacto(gluco)mannans. AGLI released galactose from melibiose and raffinose-family oligosaccharides brit tile amount of galactose released was decreased from 96% to 35% by the increasing chain length of the substrate from raffinose to verbascose. It was able to release galactose linked to the nonreducing end and less efficiently to the internal residues of the galactomanno-oligomers. AGLI was able to hydrolyze 60-92% of galactose from polymer ic galacto(gluco)mannans alone but its action was facilitated by mannanase and beta-mannosidase. rn addition, it was able to release about 10% of the galactose from softwood kraft pulp alone and about 22% in combination with mannanase. AGLII was highly specific torc ard small galactose-containing oligosaccharides in which the galactose is linked to the nonreducing end of the substrate. it released 90-100% of galactose present in melibiose, raffinose, stachyose, and verbascose; however, it was able to degrade polymeric substrates only in combination with mannanase and beta-mannosidase. AGLIII had only low activity toward the oligomeric substrates tested. It was able to release some galactose from the polymeric galacto(gluco)mannans alone, but its action was clearly enhanced br the backbone degrading enzymes.