In the central nervous system, glutamate transporters terminate the actions of this neurotransmitter by concentrating it into cells surrounding the synapse by a process involving sodium and proton cotransport followed by countertransport of potassium. These transporters contain two oppositely oriented helical hairpins 1 and 2. Hairpin 1 originates from the cytoplasm, but its tip is close to that of hairpin 2, which enters the transporter's lumen from the extracellular side. Here we address the question of whether hairpin 1 and/or domains surrounding it undergo conformational changes during the transport cycle. Therefore, we probed the reactivity of cysteines introduced into hairpin 1 and the cytoplasmic ends of transmembrane domains 6, 7, and 8 of the GLT-1 transporter to membrane-permeant N-ethylmaleimide. In each domain, except for transmembrane domain 6, cysteine mutants were found in which the inhibition of d-[(3)H]aspartate transport by the sulfhydryl reagent was increased when external sodium was replaced by potassium, a condition expected to increase the proportion of cytoplasmic-facing transporters. Conversely, the nontransportable blocker kainate protected against the inhibition in several of these mutants, presumably by locking the transporter in an outward-facing conformation. Moreover, external potassium decreased the oxidative cross-linking of two cysteines, each introduced at the tip of each hairpin. Our results are consistent with a model based on the crystal structure of an archeal homolog. According to this model, the inward movement of hairpin 1 results in the opening of a pathway between the binding pocket and the cytoplasm, lined by parts of transmembrane domains 7 and 8.