Basolateral uptake of organic anions in renal proximal tubule cells is indirectly coupled to the Na(+) gradient through Na(+)-dicarboxylate cotransport and organic anion/dicarboxylate exchange. One member of the organic anion transporter (OAT) family, Oat1, is expressed in the proximal tubule and is an organic anion/dicarboxylate exchanger. However, a second organic anion carrier, Oat3, is also highly expressed in the renal proximal tubule, but its mechanism is unclear. Thus we have assessed Oat3 function in Xenopus laevis oocytes and rat renal cortical slices. Probenecid-sensitive uptake of p-aminohippurate (PAH, an Oat1 and Oat3 substrate) and estrone sulfate (ES, an Oat3 substrate) in rat Oat3-expressing oocytes was significantly trans-stimulated by preloading the oocytes with the dicarboxylate glutarate (GA). GA stimulation of ES transport by oocytes coexpressing rabbit Na(+)-dicarboxylate cotransporter 1 and rat Oat3 was significantly inhibited when the preloading medium contained Li(+) or methylsuccinate (MS) or when Na(+) was absent. All these treatments inhibit the Na(+)-dicarboxylate cotransporter, but not rat Oat3. Li(+), MS, and Na(+) removal had no effect when applied during the ES uptake step, rather than during the GA preloading step. Concentrative ES uptake in rat renal cortical slices was also demonstrated to be probenecid and Na(+) sensitive. Accumulation of ES was stimulated by GA, and this stimulation was completely blocked by probenecid, Li(+), MS, taurocholate, and removal of Na(+). Thus Oat3 functions as an organic anion/dicarboxylate exchanger that couples organic anion uptake indirectly to the Na(+) gradient.