Na+/H+ exchange was characterized in the ileum of human brush border membrane vesicles. We have used a well-validated technique for preparing ileal brush border membranes from human cadaveric small intestine. Sodium was transported into the intravesicular space as evident by an osmolality study, which showed negligible binding to the external surface of the vesicles. The presence of an outwardly directed pH gradient stimulated Na+ uptake compared with the no pH gradient condition. Maximal uptake occurred after 5 min with a threefold overshoot over equilibrium. The rate of Na+ uptake was dependent on the extent of the pH gradient generated across the vesicles. Amiloride at 10(-4) M concentration inhibited the initial rate of Na+ uptake by greater than 90% and prevented stimulation of Na+ uptake in the presence of an outwardly directed pH gradient. The imposition of negative membrane potential did not enhance Na+ uptake compared with voltage clamp condition under both pH gradient and no pH gradient conditions, indicating an electroneutral process. Kinetic analysis of the Na+/H+ exchanger at 5 s showed a maximum velocity of 23.2 +/- 0.3 nmol/mg protein.5 s and a Michaelis constant of 14.5 +/- 5.2 mM. These studies demonstrate the presence of a Na+/H+ exchanger in human ileal brush border membrane vesicles.