Intracellular pH (pHin) affects vascular smooth muscle function, but the mechanisms that control pHin in this tissue are not well understood. These studies were performed to determine whether sarcolemmal vesicles from bovine superior mesenteric artery (SMA) contain a Na(+)-independent Cl(-)-HCO3- exchanger and, if so, to determine its sensitivity to membrane voltage and inhibitors. 36Cl- was taken up by vesicles into an osmotically active intravesicular space. In Na(+)-free media, an outwardly or inwardly directed HCO3- gradient stimulated 36Cl- transport in the opposite direction. An outwardly directed unlabeled Cl- gradient stimulated 36Cl- uptake by a mechanism that was inhibited by external HCO3-. HCO3- or Cl- gradient-stimulated 36Cl- uptake was not due to voltage coupling between ions. In the nominal absence of HCO3-, a threefold outwardly directed OH- gradient did not affect 36Cl- uptake. Total 36Cl- uptake was stimulated by an inside-positive voltage, but the HCO3- gradient-stimulated component of 36Cl- uptake was insensitive to a change in membrane voltage. Finally, HCO3- gradient-stimulated 36Cl- uptake was inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) and furosemide, with 50% inhibitory concentration values equalling approximately 1.0 and 0.5 mM, respectively. These data indicate that sarcolemmal vesicles from bovine SMA contain a Na(+)-independent Cl(-)-HCO3- exchanger. This transport system is probably electroneutral and is inhibitable by DIDS and furosemide. A conductive pathway for Cl- is present in the vesicles, but Cl(-)-OH- exchange activity was not observed.