The glutamate transporter EAAC1 (excitatory amino acid carrier 1) catalyzes the co-transport of 3 Na+ and 1 glutamate molecule into the cell. Na+ binding to the glutamate-free transporter generates a high-affinity binding site for glutamate and is, thus, required for transport. However, it is unknown which amino acid residues in the transmembrane segment contribute to controlling the affinity of the transporter for Na+ . Here, we have determined the affinity of transporters with mutations to specific amino acids in transmembrane helices TM3, TM7, and TM8 for sodium ions. We found that mutations of several conserved residues in TM3 and TM7, including N365, D367, and T101 dramatically reduce the affinity of EAAC1 for Na+ interaction with the glutamate-free transporter. The side chains of these residues are close in space in a homology model of EAAC1 generated form the crystal structure of the bacterial homologue GltPh. In contrast, mutations of E373 and D454, which are not close in space to N365, D367 and T101, do not affect the affinity for Na+ , although they impair steady- state glutamate uptake. Mutation to another amino acid residue, D439 in TM8, reduces mainly the affinity of the glutamate-bound form of the transporter for Na+ . Together, our results suggest the existence of at least two distinct Na+ binding sites on EAAC1, which are differentially affected by mutations targeted to different regions of the EAAC1 sequence.