A series of experiments were performed to demonstrate the presence of and characterize the Na-H exchanger on rabbit ileal brush border with a vesicle preparation. An outwardly directed proton gradient (pH 5.5 inside, pH 7.5 outside) stimulated Na uptake, and a fourfold "overshoot" was observed. Conversely, an inwardly directed proton gradient (pH 7.5 inside, pH 5.5 outside) inhibited Na uptake. This stimulation/inhibition of Na uptake could not be accounted for by a proton diffusion potential, because Na uptake was found to be potential insensitive. Amiloride and harmaline inhibited pH-stimulated Na uptake, but other transport inhibitors (acetazolamide, DIDS, SITS, furosemide, and bumetanide) had no effect. Amiloride also inhibited Na efflux in the presence and absence of a pH gradient. Proton gradient-stimulated Na uptake was saturable with a Km of 16.2 mM and a Vmax of 129 nmol X min-1 X mg protein-1. Tetramethylammonium did not affect pH-stimulated Na uptake, but other cations tested inhibited Na uptake, with NH4+ and Li+ causing greater inhibition than K+ or Cs+. Using the fluorescent probe acridine orange, an inwardly directed Na gradient was shown to stimulate proton efflux from the vesicles and an outwardly directed Na gradient stimulated proton influx.