The hydroxylsine-linked disaccharide unit, 2-O-alpha-D-glucopyranosyl-O-beta-D-galactopyranosyl-hydroxylysine (Glc-Gal-Hyl), prepared from collagens, was hydrolyzed by a glucohydrolase present in rat spleens and lungs. This disaccharide unit was scarcely hydrolyzed by homogenates of intestines, livers, and kidneys, which had a high alpha-D-glucosidase activity for neutral glucosides. The Glc-Gal-Hyl glucohydrolase was purified from rat spleens by affinity chromatography and gel filtration to the extent that sodium dodecyl sulfate/polyacrylamide gel electrophoresis gave a single band stained by Coomassie blue G-250. This purified glucohydrolase had a pH optimum around 5.8, and the Michaelis constant was 5.9 mM when Glc-Gal-Hyl was used as a substrate. This enzyme did not hydrolyze neutral glucosides. It is concluded that this Glc-Gal-Hyl glucohydrolase is responsible for catabolism of the hydroxylsine-linked disaccharide unit derived from collagens in mammals.