This paper provides the results of studies which characterized conductive 36Cl- flux in basolaterally enriched membrane vesicles prepared from rabbit renal outer medulla. Conductive 36Cl- uptake was studied under two different experimental conditions. In the first, 36Cl- flux was driven by an inside positive voltage created with oppositely directed Cl- and gluconate gradients. In the second, an inwardly direct K+ gradient was used to drive 36Cl- uptake. By these two methods, voltage-sensitive 36Cl- uptake was shown to comprise about 45 and 65%, respectively, of the initial rates of total 36Cl- flux. Separate paired studies demonstrated that the conductive 36Cl- uptake was inhibited by the Cl- channel blocker diphenylamine-2-carboxylate (DPC) with an IC50 for DPC of 154 microM. The voltage-dependent 36Cl- uptake had an activation energy of 6.4 kcal/mole. This 36Cl- conductance had an anion selectivity sequence of I- greater than Cl- greater than or equal to NO3- much greater than gluconate.