It is not known if the diabetes-related reduction in blood-brain barrier (BBB) transport of glucose is due to a change in the functional capacity of transporters or to an as yet unidentified mechanism occurring at the plasma membrane or cytoplasm. To increase our understanding of this problem, the cerebral blood flow, the brain uptake index (BUI) of 3-O-methyl glucose and the concentration of 3H-cytochalasin B binding sites were determined in diabetic rats and diabetic rats treated with insulin. The BUI of 3-O-methyl glucose was significantly reduced (less than 0.001) in diabetic rats (32.7 +/- 1.2%) compared to control rats (41.9 +/- 1.0%). This change could not be attributed to an alteration in cerebral blood flow or to a non-specific change in BBB permeability. Normalization of blood glucose with insulin therapy corrected the BUI measurements in diabetic rats (42.2 +/- 1.4%). The level of measurable glucose transporters measured with 3H-cytochalasin B binding assay did not appear to be reduced in the diabetic brain microvessels. The data indicate that the reduced brain uptake of glucose in chronic hyperglycemia can occur in the absence of a change in glucose transporter concentration.