We examined the effect of amiloride on Na+-H+ exchange in rabbit renal cortical microvillus membrane vesicles. Amiloride inhibited both the uphill Na+ accumulation induced by imposition of a transmembrane Hin+ greater than Hout+ gradient and the uphill H+ efflux induced by imposition of a Naout+ greater than Nain+ gradient. The inhibitory effect of amiloride on Na+ influx was rapidly reversible and fully competitive (Ki 3.0 X 10(-5) M amiloride, KT 6.3 mM Na+). In addition, amiloride inhibited the efflux of Na+ from vesicles preloaded with Na+. However, the diuretic did not inhibit such other Na+-coupled transport processes as Na+-glucose ad Na+-alanine cotransport. These findings suggest that amiloride is a reversible, selective, competitive inhibitor for the Na+ site of the renal microvillus membrane Na+-H+ exchanger. Amiloride may, therefore, be useful as an experimental tool for investigating the role of Na+-H+ exchange in mediating proximal tubular acidification.