Removal of glutamate from the synaptic cleft is carried out by transporter molecules located in presynaptic nerve terminals and fine glial processes surrounding the cleft. Three such transporters, which are approximately 55% identical to each other, have recently been cloned. They catalyze electrogenic transport of this neurotransmitter, which is coupled to the fluxes of three ions: sodium, potassium, and protons (or hydroxyl). One of these transporters, GLT-1, contains 573 amino acids and 6-10 putative membrane-spanning alpha-helices. These helices contain only two positively charged amino acid residues (lysine 298 and histidine 326) that are fully conserved in the glutamate transporters and two related neutral amino acid transporters. Using site-directed mutagenesis we have investigated the role of these residues, each of which was replaced by small hydrophilic as well as by positively charged amino acids. Expression of all replacement mutants at the histidine 326 position reveals that they are severely impaired in sodium-dependent glutamate transport. On the other hand, mutations at lysine 298 retain significant activity, especially if a positively charged amino acid replaces the lysine. After prelabeling of the proteins with [35S]methionine, immunoprecipitation of all mutant transporters indicates that their expression levels are similar to those of wild type. Reconstitution experiments, aimed to reveal the activity of transporter molecules not located in the plasma membrane, indicate that the lowered activity of the K298T and K298N transporters in intact cells is partly due to a targeting defect. Histidine residue 326 appears to be required for the intrinsic activity of the transporter. As histidine residues have been implicated in the mechanism of H+ transport in several systems, we propose that histidine 326 may be involved in the proton translocation accompanying sodium- and potassium-coupled glutamate transport.