Plasma-membrane glutamate transporters of the excitatory amino acid transporter (EAAT) family are important for maintaining a low glutamate concentration in the extracellular space of the mammalian brain. Glutamate is believed to be transported in its negatively charged form and energetically driven by the cotransport of three sodium ions, at least two of which are bound within the dielectric of the membrane. It was hypothesized that binding of substrates and competitive inhibitors is also electrogenic because the binding site is located near the center of the membrane. To test this hypothesis, we rapidly applied a low-affinity competitive inhibitor, kainate, to the glutamate transporter subtype EAAT2, resulting in outward transient current caused by movement of net negative charge of the inhibitor into the low dielectric of the protein/membrane. Consistent with these data, rate constants for inhibitor dissociation and binding were also voltage dependent. Our results are supported by electrostatic calculations and molecular dynamics simulations of spontaneous substrate dissociation, showing that the substrate and inhibitor binding site is located within the membrane environment of low dielectric constant. Charge movement caused by binding of negatively charged amino acid substrate is compensated by the charge of cotransported Na+ ion(s), thus preventing inhibition of substrate binding at negative membrane potentials. This charge compensation mechanism may be relevant for other Na+-driven transporters which recognize negatively charged substrates.