The transport of urate and p-aminohippurate (PAH) was evaluated in brush border membrane vesicles from the rat renal cortex. The binding of urate to the membranes was 6% of total uptake and no conversion of urate to allantoin was detected. The binding of PAH to the membranes was 24% of total uptake. In the presence of an outwardly directed hydroxyl ion gradient (pHi = 7.5, pHo = 6.0), the uptake of urate and PAH was stimulated relative to the absence of a hydroxyl ion gradient (pHi = pHo = 7.5) and the influx of urate resulted in a transient overshoot of the equilibrium value. The hydroxyl ion gradient-stimulated uptake of urate and PAH was not solely due to a change in membrane potential. Probenecid, DIDS, furosemide, and pyrazinoate inhibited the hydroxyl ion gradient-stimulated uptake of urate and PAH in a dose-dependent manner. The uptake of [14C]urate and [3H]PAH could be cis-inhibited and trans-stimulated by either unlabeled urate or PAH. In the presence of an outwardly directed bicarbonate gradient and 10% CO2 (outside HCO-3 = 5.4 mM, inside HCO-3 = 54 mM, pHo = 6.5, pHi = 7.5), the initial rate of urate uptake was faster and the initial rate of urate efflux was slower compared with vesicles that had the same pH gradient without bicarbonate or CO2. The effects of bicarbonate gradients on organic anion transport were not dependent on diffusion potentials. Finally, 100 mM extravesicular Na+, K+, Li+, or Cs+ did not affect urate or PAH uptake. These results indicate that brush border membrane vesicles from the rat kidney contain an anion exchange transport system with affinity for urate, PAH, hydroxyl ions, and bicarbonate. In addition there is no evidence for a sodium-urate or sodium-PAH cotransport mechanism in these membranes.