The intracellular pH in animal cells is generally maintained at a higher level than would be expected if H⁺ were passively distributed across the plasma membrane¹. In a wide variety of cells including sea urchin eggs², skeletal muscle³, renal and intestinal epithelial cells^4–6, and neuroblastoma cells⁷, plasma membrane Na⁺–H⁺ exchangers mediate the uphill extrusion of H⁺ coupled to, and thus energized by, the downhill entry of Na⁺. Plasma membrane vesicles isolated from the luminal (microvillus, brush border) surface of renal proximal tubular cells possess a Na⁺–H⁺ exchanger^4,5 that seems to be representative of the Na⁺–H⁺ exchangers found in other tissues. For example, the renal microvillus membrane Na⁺–H⁺ exchanger, like other N⁺–H⁺ exchangers, mediates electroneutral cation exchange^4,5, is sensitive to inhibition by the diuretic drug amiloride^5,8, and has affinity for Li⁺ in addition to Na⁺ and H⁺ (refs 5, 9). Here we have examined the effect of internal H⁺ on the activity of the Na⁺–H⁺ exchanger in renal microvillus membrane vesicles. Our results suggest that internal H⁺, independent of its role as a substrate for exchange with external Na⁺, has an important modifier role as an allosteric activator of the Na⁺–H⁺ exchanger. Allosteric behaviour with respect to internal H⁺ is a property that would enhance the ability of plasma membrane Na⁺–H⁺ exchangers to extrude intracellular acid loads and thereby contribute to the regulation of intracellular pH.