Glutamate is the major excitatory neurotransmitter in mammalian central nervous system. After its release from glutamatergic nerve terminals, glutamate is quickly taken up into glial and neuronal cells by glutamate transporters belonging to the "excitatory amino acid transporter" family. In recent years, various EAAT paralogs have been identified in prokaryotes and shown to exhibit a variety of transport stoichiometries. Whereas EAATs transport 3 Na⁺, 1 H⁺, and 1 glutamate in countertransport with one K⁺, there are bacterial paralogs that only transport glutamate stoichiometrically coupled to H⁺, or aspartate coupled to Na⁺. We reasoned that the simplicity of the transport stoichiometry of bacterial transporters might allow transplantation of transport mechanisms from one isoform to the other and thus provide insights into molecular determinants of coupled transport. We studied radiotracer flux accumulation by purified and reconstituted glutamate transporters from E. coli (ecgltP). Glutamate transport by ecgltP is independent of Na⁺ and K⁺, in marked contrast to mammalian EAATs and bacterial glutamate transporter from Pyrococcus horikoshii (Glt_Ph). ecgltP transports H⁺ and glutamate, and voltage dependence of radiotracer flux accumulation allowed determination of a transport stoichiometry of one glutamate coupled to three protons. Although transport of ecgltP is independent of Na⁺, ecgltP lacks only one of the candidate sodium binding sites previously identified in prokaryotic (Boudker et al. (2007) Nature 445, 387–393) or mammalian (Tao et al. (2006) J Biol Chem 281, 10263–10272; Tao & Grewer (2007) J Gen Physiol 129, 331–344) isoforms, an aspartate at position 401. We tried to insert the missing sodium binding site into ecgltP by substituting asparagine by aspartate at this position. Similar to WT ecgltP, transport by N401D ecgltP mutant is voltage-dependent and coupled to intra- and extravesicular pH. The voltage dependence of radiotracer flux accumulation reveals that only two positive charges are co-transported with glutamate by N401D ecgltP. In contrast to our expectation, glutamate uptake of N401D ecgltP is not sodium-dependent. We conclude that N401D ecgltP transports 2 H⁺ and 1 glutamate, indicating that coupling of glutamate and Na⁺ transport can not be transplanted from Glt_Ph to ecgltP by inserting a single negative charge and that the molecular requirements for coupled sodium-glutamate transport are more complex than currently thought. Moreover, N401 plays an additional role in defining the number of co-transported protons in ecgltP.