The nhaB gene which codes for Na+/H+ antiporter activity in Escherichia coli was recently cloned (Pinner, E., Padan, E., and Schuldiner, S. (1992) J. Biol. Chem. 267, 11064-11068). In order to elucidate the role of nhaB in Na+ and H+ ions physiology and its interaction with nhaA, we generated mutants in which the chromosomal gene has been inactivated by insertion/deletion. A mutant devoid of both nhaA and nhaB is extremely sensitive to Na+ and Li+ at all pH values, and membranes prepared from this strain show no Na+/H+ antiporter activity. As opposed with the delta nhaA mutant which contains NhaB, the pH independent Na+/H+ antiporter (Padan, E., Maisler, N., Taglicht, D., Karpel, R., and Schuldiner, S. (1989) J. Biol. Chem. 264, 20297-20302), the delta nhaB mutant, containing NhaA, shows Na+/H+ antiporter activity highly dependent on pH. nhaB, in the absence of nhaA, confers a certain tolerance to Na+ which decreases with increasing pH. In the absence of NhaB, NhaA alone confers complete halotolerance under all conditions tested. However, when grown on agar in minimal medium on substrates which are symported with Na+ (proline, serine, and glutamate) at pH 6 and at low Na+ concentrations (< 10 mM), delta nhaB grows slower than the wild type and its Na+ dependent transport of glutamate and proline is markedly inhibited. Since both of these defects of the delta nhaB strain are alleviated upon transformation of the mutant with multicopy plasmid bearing nhaA, we conclude that nhaB is crucial when the level of NhaA activity is growth limiting, when nhaA is not sufficiently induced, and/or when NhaA is not activated.