l‐Glutamate transport by the H+‐glutamate and Na+‐glutamate symport proteins of Escherichia coli K‐12 (GltPEc and GltSEc, respectively) and the Na+‐H+‐glutamate symport proteins of Bacillus stearothermophilus (GltTBs) and Bacillus caldotenax (GltTBc) was studied in membrane vesicles derived from cells in which the proteins were either homologously or heterologously expressed. Substrate and inhibitor specificity studies indicate that GltPEc, GltTBs and GltTBc fall into the same group of transporters, whereas GltSEc is distinctly different from the others. Also, the cation specificity of GltSEc is different; GltSEc transported l‐glutamate with (at least) two Na+, whereas GltPEc, GltTBs and GltTBc catalysed an electrogenic symport of l‐glutamate with ≥two H+, i.e. when the proteins were expressed in E. coli Surprisingly studies in membrane vesicles of B. stearothermophilus and B. caldotenax indicated a Na+‐H+‐l‐glutamate symport for both GltTBs and GltTBc. The Na+ dependency of the GltT transporters in the Bacillus strains increased with temperature. These observations suggest that the conformation of the transport proteins in the E. coli and the Bacillus membranes differs, which influences the coupling ion selectivity.