Diabetes was induced in male Lewis rats by a single injection of streptozocin (50 mg/kg body wt ip). After 10-14 days, diabetic and age- and sex-matched control animals were killed, and their proximal small intestines were removed. Villus-tip, mid-villus, and lower-villus enterocytes were harvested from each group with a method that combined divalent cation chelation with mild mechanical dissociation. These fractions were used as starting material to prepare brush-border membrane vesicles. Preparations from each of these fractions were then analyzed and compared with respect to their Na(+)-gradient-dependent and Na(+)-independent D-glucose transport, lipid fluidity, and lipid composition. The results of these experiments demonstrated that 1) maximum rates of Na(+)-gradient-dependent D-glucose transport (Vmax) were greatest in membrane vesicles prepared from mature cells (villus tip and mid villus) of control rats; 2) the glucose concentration producing half-maximal rates of transport (Km), however, was significantly lower in lower-villus membrane vesicles of control rats, suggesting that a distinct glucose transporter existed in the membranes of these relatively immature enterocytes; 3) Na(+)-gradient-dependent, but not Na(+)-independent, D-glucose uptake was greater in diabetic membrane vesicles prepared from mid-villus and lower-villus fractions but not in vesicles prepared from villus-tip cells; and 4) no obvious relationship between alterations in membrane lipid fluidity and enhanced uptake of Na(+)-gradient-dependent D-glucose by these transporter(s) could be established in this experimental model of acute diabetes mellitus.