: The transport of glucose across the blood‐brain barrier (BBB) is mediated by the high molecular mass (55‐kDa) isoform of the GLUT1 glucose transporter protein. In this study we have utilized the tritiated, impermeant photolabel 2‐N‐[4‐(1‐azi‐2,2,2‐trifluoroethyl)[2‐³H]propyl]‐1,3‐bis(d‐mannose‐4‐yloxy)‐2‐propylamine to develop a technique to specifically measure the concentration of GLUT1 glucose transporters on the luminal surface of the endothelial cells of the BBB. We have combined this methodology with measurements of BBB glucose transport and immunoblot analysis of isolated brain microvessels for labeled luminal GLUT1 and total GLUT1 to reevaluate the effects of chronic hypoglycemia and diabetic hyperglycemia on transendothelial glucose transport in the rat. Hypoglycemia was induced with continuous‐release insulin pellets (6 U/day) for a 12‐ to 14‐day duration ; diabetes was induced by streptozotocin (65 mg/kg i.p.) for a 14‐ to 21‐day duration. Hypoglycemia resulted in 25‐45% increases in regional BBB permeability‐surface area (PA) values for d‐[¹⁴C]glucose uptake, when measured at identical glucose concentration using the in situ brain perfusion technique. Similarily, there was a 23 ± 4% increase in total GLUT1/mg of microvessel protein and a 52 ± 13% increase in luminal GLUT1 in hypoglycemic animals, suggesting that both increased GLUT1 synthesis and a redistribution to favor luminal transporters account for the enhanced uptake. A corresponding (twofold) increase in cortical GLUT1 mRNA was observed by in situ hybridization. In contrast, no significant changes were observed in regional brain glucose uptake PA, total microvessel 55‐kDa GLUT1, or luminal GLUT1 concentrations in hyperglycemic rats. There was, however, a 30‐40% increase in total cortical GLUT1 mRNA expression, with a 96% increase in the microvessels. Neither condition altered the levels of GLUT3 mRNA or protein expression. These results show that hypoglycemia, but not hyperglycemia, alters glucose transport activity at the BBB and that these changes in transport activity result from both an overall increase in total BBB GLUT1 and an increased transporter concentration at the luminal surface.