We investigated the transmembrane topology of the glutamate receptor GluR1 by introducing N-glycosylation sites as reporter sites for an extracellular location of the respective site. Our data show that the N-terminus is extracellular, whereas the C-terminus is intracellular. Most importantly, we found only three transmembrane domains (designated TMD A, TMD B, and TMD C), which correspond to the previously proposed TMDs I, III, and IV, respectively. Contrary to earlier models, the putative channel-lining hydrophobic domain TMD II does not span the membrane, but either lies in close proximity to the intracellular face of the plasma membrane or loops into the membrane without transversing it. Furthermore, the region between TMDs III and IV, in previous models believed to be intracellular, is an entirely extracellular domain.