This study describes the effects of streptozocin (STZ)-induced diabetes mellitus on the glucose-transporter system of the rat blood-brain barrier. Subcellular membrane fractions, i.e., plasma membranes and high- and low-density microsomes, were prepared from isolated brain microvessels derived from control and diabetic animals. The number of glucose transporters in each of the membrane fractions from both control and diabetic animals was determined by the D-glucose-inhibitable cytochalasin B-binding assay. The total number of glucose transporters was decreased by 43% in STZ-treated rats compared with controls (35 vs. 115 pmol/mg protein; P less than .05). The glucose-transporter number in plasma membranes was decreased by 50%, in high-density microsomes by 38%, and in low-density microsomes by 45%. Incubation of isolated microvessels from control animals with 7 microM insulin for 30 min at 37 degrees C led to a cycloheximide-sensitive 27% increase (P less than .05) in the number of transporters in high-density microsomes. This insulin effect was significantly diminished to 15% in the diabetic animals (P less than .05). In conclusion, STZ-induced diabetes decreases the glucose-transporter number in all subcellular membrane fractions derived from isolated rat brain microvessels, and the insulin-induced increase in de novo synthesis of glucose transporters in brain microvessels is diminished in these chronically diabetic animals.