We have previously shown that HPNhaA (Helicobacter pylori Na+/H+ antiporter) forms an oligomer in a native membrane of Escherichia coli, and conformational changes of oligomer occur between monomers of the oligomer during ion transport. In the present study, we use Blue-native PAGE to show that HPNhaA forms a dimer. Cysteine-scanning mutagenesis of residues 55-61 in a putative beta-sheet region of loop1 and subsequent functional analyses revealed that the Q58C mutation resulted in an intermolecular disulfide bond. G56C, I59C and G60C were found to be cross-linked by bifunctional cross-linkers. Furthermore, the Q58E mutant did not form a dimer, possibly due to electrostatic repulsion between monomers. These results imply that Gln-58 and the flanking sequence in the putative beta-sheet of the monomer are located close to the identical residues in the dimer. The Q58C mutant of NhaA was almost inactive under non-reducing conditions, and activity was restored under reducing conditions. This result showed that cross-linking at the dimer interface reduces transporter activity by interfering with the flexible association between the monomers. A mutant HPNhaA protein with three amino acid substitutions at residues 57-59 did not form a dimer, and yet was active, indicating that the monomer is functional.