The transport of urate was evaluated in brush border membrane vesicles isolated from the dog renal cortex. It was previously shown that uphill urate and p-aminohippurate (PAH) uptake into these membrane vesicles results from imposing an outwardly directed OH- gradient. In the present study, the OH- gradient-stimulated uptake of urate was inhibited by external Cl-. In the absence of OH- gradients, imposing an outwardly directed Cl- gradient induced the transient accumulation of urate against its concentration gradient (overshoot), whereas imposing an inwardly directed Cl- gradient induced the transient uphill efflux of urate (undershoot). The effects of Cl- gradients on urate transport persisted when the diffusion potentials caused by the Cl- gradients were negated by superimposing K+ gradients in the presence of the K+ ionophore valinomycin. The dose-response curves for three different inhibitors were identical for the OH- and Cl- gradient-stimulated modes of urate uptake. The uptake of PAH was also stimulated by imposing an outwardly directed Cl- gradient. The dose-response curves for probenecid inhibition were identical for the OH- and Cl- gradient-stimulated modes of PAH uptake. Finally, the existence of a Na+ cotransport pathway for urate or PAH could not be demonstrated. We conclude that the principal mechanism for urate transport in dog renal microvillus membrane vesicles is via an anion exchanger with affinity for urate, PAH, OH-, and Cl-. This anion exchanger may play an important role in mediating organic anion reabsorption and secretion in the proximal tubule.