Glucosyltransferase (UDPglucose: galactosylhydroxylysine-basement membrane glucosyltransferase), an enzyme specifically involved in collagen synthesis, was measured in various kidney fractions of normal, diabetic and underfed rats, using as basis the incorporation of radioactivity into protein during incubation with UDP[U-14C]glucose and alkali-soluble fetal calf-skin collagen. Three criteria of enzyme activity were compared: A, total radioactivity of the washed protein precipitate; B, this figure minus activity incorporated in the absence of the collagen acceptor; and C, radioactivity incorporated into the mixed amino acid fraction, collected by elution with dilute NH4OH from a Dowex 50 resin column after alkaline hydrolysis of the protein. Method A was found satisfactory using whole medulla or isolated glomeruli, since the average proportions of total protein radioactivity recovered in the NH3 fraction were 0.81 and 0.87, respectively, and the deviations were small. There was a larger and variable proportion of nonspecific incorporation using whole cortex. Incubation of a control set of sample without added collagen was found to be unnecessary (Method B). Per mg protein, medulla and glomeruli had more enzyme than did whole cortex. In diabetes, activity was enhanced in the 10,000 X g supernatant fraction of cortex, as previously reported. However, the increase associated with diabetes was even more consistent in the medulla, averaging 3-fold in the 10,000 X g pellet fraction. No increase was found in isolated glomeruli in diabetes. Also, no increase was seen in the kidneys of non-diabetic rats with body weight similar to that of the diabetics.