Abstract The tissue distribution in the rat of the glucosyl- and galactosyltransferases involved in the biosynthesis of the hydroxylysine-linked disaccharide (2-O-α-d-glucopyranosyl-d-galactose) and monosaccharide (galactose) units has been investigated. High levels of these enzymes have been found in tissues rich in collagen and basement membranes, including cartilage, uterus, skin, spleen, lung, kidney cortex, and thyroid. Low levels were present in liver, while brain and heart were almost devoid of these enzymes. The relative amounts of these two glycosyltransferases varied, with cartilage, skin, spleen, and lung having similar levels of the two enzymes, while in kidney, thyroid, and uterus there was more of the glucosyl-than the galactosyltransferase. The two enzymes could be purified from cartilage by Bio-Gel A-1.5m filtration in a manner similar to that previously used for the kidney cortex. The activity of the two glycosyltransferases varied with age in a parallel manner, reaching a maximum at about 9 days after birth, and falling off thereafter to reach levels of about 10% of the maximum in the mature animals. A marked increase in the level of the glucosyltransferase of the uterus was observed in pregnancy, with a doubling of the specific activity and a 16-fold increase in the total activity. The enzyme decreased toward normal in the postpartum state. Measurement of the glucosyltransferase in the renal cortex of alloxan diabetic rats indicated a highly significant increase in activity over age-matched normals, and was believed to reflect an increased synthesis of basement membrane in the diabetic kidney. Differential centrifugation of kidney cortex homogenates in buffered sucrose indicated that both the glucosyl- and galactosyltransferase were present primarily in a rapidly sedimenting particle fraction (650 and 2000 x g), as well as in the high speed supernatant (100,000 x g). The amount of enzyme in the supernatant varied with the medium used for homogenization.