Glucose is the primary metabolic fuel for the mammalian brain, and a continuous supply is required to maintain normal CNS function. The transport of glucose across the blood–brain barrier (BBB) into the brain is mediated by the facilitative glucose transporter GLUT-1. Prior studies (Simpson et al.  J Biol Chem 276:12725–12729) had revealed that the conformations of the GLUT-1 transporter were different in luminal (blood facing) and abluminal (brain facing) membranes of bovine cerebral endothelial cells, based on differential antibody recognition. This study has extended these observations and, by using a combination of 2D-PAGE/Western blotting and immunogold electron microscopy, determined that these different conformations are exhibited in vivo and arise from differential phosphorylation of GLUT-1 and not from alternative splicing or altered O- or N-linked glycosylation. © 2011 Wiley-Liss, Inc.