We investigated the mechanism of Cl- transport in microvillus membrane vesicles isolated from Necturus kidneys. Cl- influx was insensitive to changes in membrane potential induced by K+ gradients and the K+ ionophore valinomycin, arguing against conductive Cl- transport. Inward gradients of Na+ or Na+ + K+ did not stimulate initial Cl- influx, arguing against direct Na+-Cl- or Na+-K+-Cl- cotransport. External Cl-, HCO3-, and NO3- each stimulated 36Cl efflux and inhibited 36Cl uptake, indicating anion exchange. Outward HCO3- gradients but not OH- gradients stimulated 36Cl influx, consistent with Cl- -HCO3- exchange. Cl- transport via anion exchange was inhibited by furosemide, bumetanide, and disulfonic stilbenes, but not by acetazolamide. External halides stimulated 36Cl efflux (Cl- = Br- greater than I- greater than F-) but the organic anions lactate, p-aminohippurate, and urate did not. Amiloride-sensitive Na+-H+ exchange was demonstrated. Finally, in the presence of a CO2/HCO3 buffer system, imposing an inward Na+ gradient caused a time-delayed stimulation of 36Cl uptake, consistent with indirect coupling of Na+-H+ and Cl- -HCO3- exchangers. We conclude that the parallel operation of Na+-H+ and Cl- -HCO3- exchangers rather than direct cotransport may account for the Na+-coupled uphill Cl- entry previously observed in the intact proximal tubular cell of Necturus.