A H+ gradient (intravesicular greater than extravesicular), in the absence of a Na+ gradient (extravesicular greater than intravesicular) stimulated phosphate uptake by renal brush border membrane vesicles and provided the driving force to effect the transient accumulation of phosphate against its concentration gradient. The H+ gradient-dependent uptake of phosphate had an absolute requirement for Na+. The rates of uptake and peak accumulation were functions of the delta pH and the concentration of H+ in the intravesicular medium. The H+ gradient-energized Na+-phosphate cotransport system was not affected by valinomycin- or carbonyl cyanide p-fluoromethoxyphenylhydrazone-induced ion diffusion potentials. Therefore, it was independent of the membrane potential, i.e. an electroneutral process. Amiloride, which inhibited the H+-Na+ exchange reaction and prevented the efflux of H+ from the intravesicular medium, enhanced the uptake of phosphate. A model is proposed by which the H+ gradient mediates the uphill transport of phosphate. It is suggested that a similar process may operate in more physiologically intact preparations and may provide one mechanism by which acid-base balance regulates renal phosphate transport.