Abstract A glucosyltransferase has been prepared from rat kidney cortex which is involved in the assembly of the hydroxylysine-linked glucosylgalactose disaccharide units of basement membranes and collagens. This enzyme has been purified from the high speed supernatant of the kidney cortex of 10-day-old rats by ammonium sulfate fractionation and Bio-Gel filtration. It has also been obtained by ultrasonic treatment of a particulate fraction from the kidney cortex. The enzyme was found to transfer glucose from UDP-glucose to hydroxylysine-linked galactose, either in high or low molecular weight compounds. These acceptors include native collagen from varied sources, glomerular basement membrane from which the glucose had been selectively removed, peptides containing galactosylhydroxylysine or galactosylhydroxylysine itself. The Km values for native calf skin collagen and glucose-free basement membrane were found to be 1.3 and 1.6 x 10-4 m, respectively, while that for galactosylhydroxylysine-containing peptides was determined to be 1.2 x 10-3 m, indicating that the enzyme has a greater affinity for the high molecular weight acceptors. Characterization of the product indicated that the enzyme formed the naturally occurring hydroxylysine-linked disaccharide (2 - O - α - d - glucopyranosyl - d - galactose). This enzyme may be designated as a UDP-glucose:galactosylhydroxylysine-collagen (basement membrane) glucosyltransferase, its primary role in kidney cortex being to participate in basement membrane synthesis. It was found that the e-amino group of the hydroxylysine, which is vicinal to the hydroxyl group to which galactose is attached, has to be unsubstituted in order for a compound to serve as an acceptor. The enzyme was inactive toward substrates containing terminal galactose residues linked to other amino acids or sugars, including sialic acid-free thyroglobulin glycopeptides, earthworm cuticle collagen glycopeptides, or lactose. Galactosylsphingosine (psychosine), however, was found to be an acceptor for this enzyme. Glucosylgalactosylsphingosine was formed and this transfer activity was shown by competitive inhibition studies to be a function of the same enzymatic site as is involved in the formation of glucosylgalactosylhydroxylysine. This lack of specificity is believed to be caused by the similarity which exists between the glycosidic linkage region of galactosylhydroxylysine and galactosylsphingosine. Although UDP-glucose was the preferred glycosyl donor for the glucosyltransferase with a Km value of 3.1 x 10-5 m, TDP- and CDP-glucose could serve in this capacity to a limited extent. The enzyme was found to have an absolute requirement for a divalent cation, manganese being the most effective. The pH optimum was 7.4. It was shown that the glucosyltransferase, working in concert with a kidney galactosyltransferase, could effect the synthesis of the hydroxylysine-linked disaccharide unit.