The ventromedial hypothalamus (VMH) has been proposed to be a glucose sensor within the brain and appears to play a critical role in initiating the counterregulatory response to hypoglycemia. Transport of glucose across the brain capillaries and into neurons in this region is mediated by different isoforms of the sodium-independent glucose transporter gene family. The objective of the present study was to identify the specific glucose transporter isoforms present, as well as their cellular localization, within the VMH. Immunohistochemistry was performed for GLUT1, GLUT2 and GLUT4 in frozen sections of hypothalami from normal rats. GLUT1 was present on the endothelial cells of the blood-brain barrier (BBB) of the VMH. GLUT2 immunoreactivity was seen in the ependymal cells of the third ventricle and in scattered cells in the arcuate and periventricular nuclei. There was no GLUT2 expression in the VMH. The insulin-sensitive GLUT4 isoform was localized to vascular structures within the VMH. Double-labeled immunohistochemistry demonstrated co-localization of GLUT4 with GLUT1 and with the tight junction protein ZO-1 in the VMH and suggested that VMH GLUT4 expression was restricted to the BBB. The role of GLUT4 in the brain and within the VMH is unknown, but given its location on the BBB, it may participate in brain sensing of blood glucose concentrations.