Experiments with basolateral membrane vesicles prepared from rat kidney cortex were performed to study the mechanism by which p-aminohippuric acid (PAH) is taken up across the contraluminal membrane and is concentrated in proximal tubule cells. An inward Na+ gradient failed to stimulate [3H]PAH uptake compared with K+ or Li+ and did not cause intravesicular PAH accumulation above equilibrium distribution. In the absence of Na+, the dicarboxylates glutarate and suberate cis-inhibited and trans-stimulated [3H]PAH uptake, indicating a common transport system. In the presence of Na+, 10 microM glutarate in the incubation medium did not cis-inhibit, but rather stimulated [3H]PAH uptake and caused PAH accumulation above equilibrium distribution ("overshoot"). Li+ diminished this stimulation, but was without effect on [3H]PAH/PAH- and [3H]PAH/glutarate exchange. The data indicate the coexistence of a Na+ -coupled, Li+-sensitive transport system for dicarboxylates and a Li+ -insensitive PAH/dicarboxylate exchanger in the basolateral membrane. We propose that dicarboxylates are cotransported with Na+ into the cell and subsequently exchange for extracellular PAH at the basolateral membrane. PAH uptake is thereby indirectly coupled to Na+ via the Na+/dicarboxylate cotransporter.