Cl-transport was studied in microvillus membrane vesicles isolated from the rabbit renal cortex. Inwardly directed K+ gradients in the presence of the K+ ionophore valinomycin (Val) enhanced 10 mM 36Cl uptake 2.5-fold, confirming a Cl- conductive pathway. An inwardly directed H+ gradient (pHin 7.5, pHout 6.0) stimulated 10 mM Cl- uptake 1.5-fold compared with pHin = pHout = 6.0. However, this H+ gradient stimulation of Cl- uptake appeared secondary to the H+ diffusion potential rather than to Cl-OH exchange, as it was abolished by Val and K+in = K+out. Additional evidence against Cl- transport via anion exchange was the failure of an inwardly directed Cl- gradient to generate an inside-acid pH gradient as monitored by quenching of acridine orange fluorescence. Cl- influx was the same in the presence of inwardly directed gradients of Na+, K+, Cs+, Li+, and Rb+, arguing against NaCl cotransport. Finally, conductive Cl- transport was reduced by the inhibitors furosemide, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid and 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid. These data indicate the presence of an inhibitor-sensitive, conductive mode of Cl- transport but fail to demonstrate significant pathways for Cl- OH exchange or NaCl cotransport in rabbit renal microvillus membrane vesicles.