The mechanisms that control intracellular pH (pHi) in vascular smooth muscle are not fully understood. We reported that pHi in primary cultured vascular smooth muscle cells from canine femoral artery is 7.26, a value maintained via HCO3- influx by the Na(+)-dependent C1(-)-HCO3-exchanger but not via H+ efflux by the Na(+)-H+ exchanger [A. M. Kahn, E. J. Cragoe, Jr., J. C. Allen, R. D. Halligan, and H. Shelat. Am. J. Physiol 259 (Cell Physiol. 28): C134-C143, 1990]. To explain these findings, in the present study, we determined the pHi activity profile of these two transport systems. Although both were active at acidic pHi, Na(+)-H+ exchange activity was very low at and above pHi 7.0, while Na(+)-dependent C1(-)-HCO3-exchange activity maintained near-maximal activity up to pHi 7.26 but fell to undetectable levels by pHi 7.4. A Na(+)-independent C1(-)-HCO3-exchanger was present, which mediated HCO3-efflux after an acute alkaline load. The activity of this system was negligible at pHi 7.2 and was stimulated at alkaline pHi. In conclusion, the pHi of these vascular smooth muscle cells at rest is maintained via HCO3-influx by the Na(+)-dependent C1(-)-HCO3-exchanger. After acute acidic or alkaline loads, correction of pHi is mediated by activation of the normally quiescent Na(+)-H+ and Na(+)-independent C1(-)-HCO3-exchangers, respectively. All three acid-base transport systems have pHi set points that protect the cell from overcorrecting pHi after a disturbance in either direction.