The H+:Na+ exchange stoichiometry of NhaA, a sodium-proton antiporter coded by the nhaA gene of Escherichia coli, has been determined using purified NhaA protein reconstituted into sodium-loaded proteoliposomes. One approach involved measuring, in parallel experiments, the Na+ efflux and H+ influx from such proteoliposomes and calculating the stoichiometry from the ratio of these fluxes. A second approach was based on measuring the membrane potential generated by NhaA at various sodium gradients and assuming complete coupling and thermodynamic equilibrium between the membrane potential and the ion gradients. The results from both methods agree with a stoichiometry of 2 H+ exchanged for each Na+. This value is independent of pH between pH 7.2 and 8.1. These results support the suggestion that a change in the catalytic rate of NhaA rather than its stoichiometry is crucial for its role in regulation of intracellular pH in alkaline environments.