Synaptic transmission is commonly terminated by diffusion and reuptake of neurotransmitter from the synaptic cleft. Glutamate reuptake prevents neurotoxicity and sets the lower limit for the concentration of extracellular glutamate, so it is important to understand the thermodynamics of this process. Here we use voltage clamping with a pH-sensitive fluorescent dye to monitor electrical currents and pH changes associated with flux of glutamate mediated by the human neuronal glutamate transporter EAAT3. In contrast to a previous model, we find that three sodium ions and one proton are cotransported with each glutamate ion into the cell, while one potassium ion is transported out of the cell. This coupling can support a transmembrane glutamate concentration gradient ([Glu]in/[Glu]out) exceeding 10(6) under equilibrium conditions, and would allow the transporter to continue removing glutamate over a wide range of ionic conditions.