The delta pH-dependent quenching of Acridine orange was used to characterize Na+-H+ exchange and K+ and H+ conductances in brush-border membrane vesicles isolated by precipitation with either CaCl2 or MgCl2 from rat kidney cortex. A transmembrane pH difference of 2.5 units (inside acidic) was imposed and the initial rate of its dissipation was followed after injecting a puls of tetramethylammonium gluconate (control) or sodium or potassium gluconate. In membranes isolated by CaCl2, the Na+-H+ exchange was partially electroneutral (45% to 77% of the total exchange) and the rest was due to electrically coupled Na+ and H+ movements through conductive pathways in the membranes. In membranes prepared by MgCl2, the rate of total Na+-H+ exchange was about twice as high as that in membranes obtained by CaCl2 precipitation. However, total and electroneutral exchanges were equal indicating negligible electrically coupled Na+ and H+ movements in these membranes. K0.5 for Na+ in all preparations was in the same range, being in average 30 mM. Amiloride was a competitive inhibitor of Na+-H+ exchange in membranes obtained with both preparations; Ki values ranged between 0.1 and 0.58 mM. The rates of delta pH-dissipation with K+ gradients (+/- valinomycin) were by 50% to 150% higher in membranes prepared with CaCl2 than in membranes isolated with MgCl2 indicating much higher H+ and K+ conductances in membranes obtained with CaCl2. Therefore, the rate of Na+-H+ exchange as well as the conductances for various ions in the isolated brush-border membranes depend on membrane preparation.