Controversial evidence exists as to whether electroneutral Cl-OH exchange occurs across the proximal tubular brush border membrane of the mammalian kidney. To help resolve this controversy, we carried out a series of experiments using rabbit renal cortical brush border membrane vesicles to determine the effect of changes in pH on 36Cl uptake across this membrane. Reducing pH equally inside and outside of the vesicle stimulated Cl-Cl exchange as well as chloride uptake. A proton gradient (out greater than in) stimulated and caused an overshoot in unidirectional Cl uptake. All three of these processes were inhibited by 4-acetoamido-4-isothiocyano-2,2-disulfonic stilbene. Valinomycin with K+ out-in added to shunt any proton diffusion potential, minimally inhibited pH gradient-dependent Cl uptake, and carbonyl cyanide p-trifluoro-methoxyphenyl hydrazone (FCCP), added to increase the proton diffusion potential, minimally stimulated pH gradient-dependent Cl uptake. Sodium-glucose cotransport (an electrogenic system) was used to assess the efficacy of these ionophores. FCCP markedly enhanced and valinomycin markedly inhibited pH gradient stimulated sodium-glucose cotransport. Valinomycin added to vesicles with a 180/0 meq K+ out greater than in gradient stimulated less Cl uptake than a 6.0/7.5 pH out greater than in gradient. We conclude that these results provide strong evidence for the existence of an electroneutral Cl-OH exchanger or Cl-H symporter in this membrane.