The diuretic amiloride stimulates the rate of decay of a proton gradient in both native ileal brush border membrane vesicles and artificial phospholipid vesicles. The kinetics of the rate of decay of the pH gradient were studied using acridine orange in membrane vesicles equilibrated at pH 5.5 and assayed at pH 8.0. Amiloride stimulated the decay of the proton gradient in a dose-dependent fashion. In parallel experiments employing artificial phospholipid vesicles and the monovalent cation-H+ exchanger nigericin, amiloride inhibited both the initial rate and extent of nigericin-mediated 22Na+ accumulation. It is concluded from these studies in both native and artificial membrane vesicles that amiloride can act as permeant weak base thus dissipating the pH gradient necessary for Na+ accumulation via a Na+-H+ exchanger.