In the central nervous system, (Na+ + K+)-coupled glutamate transporters restrict the neurotoxicity of this transmitter and limit the duration of synaptic excitation at some synapses. The various isotransporters exhibit a particularly high homology in an extended hydrophobic domain of ill-defined topology that contains several determinants involved in ion and transmitter binding. Here, we describe the determination of the membrane topology of the cloned astroglial glutamate transporter GLT-1. A series of functional transporters containing single cysteines was engineered. Their topological disposition was determined by using a biotinylated sulfhydryl reagent. The glutamate transporter has eight transmembrane domains long enough to span the membrane as et heiices. Strikingly, between the seventh and eighth domains, a structure reminiscent of a pore loop and an outward-facing hydrophobic linker are positioned.