Endo-beta-galactosidase was purified 4400-fold from a culture filtrate of Escherichia freundii with 45% recovery. The enzyme preparation was practically free of exoglycosidases, sulfatase, and proteases. This enzyme hydrolyzed several keratan sulfates, endoglycosidically releasing oligosaccharides of various molecular sizes. Among the digestion products of the corneal keratan sulfate, the structure of a disaccharride and a tetrasaccharride were shown to be 2-acetamido-2-deoxy-6-O-sulfo-beta-D-glucosyl-(1 leads to 3)-D-galactose and 2-acetamido-2-deoxy-6-O-sulfo-beta-D-glucosyl-(1 leads to 3)-6-O-sulfo-beta-D-galactosyl-(1 leads to 4)-2-acetamido-2-deoxy-6-O-sulfo-beta-D-glucosyl-(1 leads to 3)-D-galactose, respectively. These oligosaccharide structures indicate that this enzyme specifically hydrolyzes the galactosidic bonds in which nonsulfated galactose residues participate. The enzyme could also hydrolyze a small oligosaccharide such as lacto-N-neotetraitol as follows: Gal(beta 1 leads to 4)GlcNAc(beta 1 leads to 3)Gal(beta 1 leads to 4) sorbitol leads to Gal(beta 1 leads to 4)GlcNAc(beta 1 leads to 3)Gal + sorbitol AB active blood group substance could be hydrolyzed by this enzyme only after Smith degradation. After enzymatic digestion small oligosaccharides and resistant macromolecules were produced. These findings indicate that the enzyme should be useful in studying the precise structures of keratan sulfates, related glycoproteins, and oligosaccharides.