The importance of negatively charged residues in transmembrane helices of many cation-coupled transporters has been widely demonstrated. Four Asp residues were located in the putative transmembrane helices of the Escherichia coli Na+/H+ antiporter, NhaA. We replaced each of these Asp residues by Asn in plasmid encoded nhaA and expressed these constructs in an E. coli mutant defective in both nhA and nhaB. Substitution of Asp-65 or Asp-282 (in the extramembrane region) had no effect on supporting the host mutant growth in the high NaCl- or LiCl-containing medium, and these two mutants had normal Na+/H+ and Li+/H+ antiporter activities. In contrast, substitution of Asp-133, Asp-163 or Asp-164 was detrimental to survival of the host mutant and impaired both Na+/H+ and Li+/H+ antiporter activities. These three Asp residues, conserved in the nhaA homologs from different species and which are located closely in the 3rd and 4th putative transmembrane helices, appear to play important roles in cation binding and transport.