pHois an important determinant of vascular tone in cerebral blood vessels. We investigated the effects of changes in pH. on isolated smooth muscle cells from the basilar artery of the guinea pig. Single cells contracted rapidly in response to an elevation in pHo(constant CO2), and contraction was blocked by nifedipine, suggesting a role for dihydropyridine-sensitive Ca2+channels. In whole-cell patch-clamp experiments, changes in pHo(pHo5.7–8.1, pHi7.2 with 10 mM HEPES) strongly affected the amplitude of the peak Ca2+channel current (10 mM Ba2+, +15 mV, holding potential of −55 mV), with an apparent pK of 6.9. The current-voltage curves were minimally shifted, indicating no important effect of surface charge. To separate the slowly inactivating L-type Ca2+channel current from the more rapidly inactivating B-type current, the decaying portions of inward currents from cells studied with repetitive 1-second pulses (+15 mV, holding potential of −55 mV) were fit to a two-component model. Titration curves for the L-type and B-type currents indicated maximum increases by factors of 3.65 and 1.28 at alkaline pHoand gave apparent pK values of 7.71 and 6.47 (Hill coefficient unity). The time constant of inactivation for the B-type current at +15 mV was little affected by pHo, whereas that for the L-type current increased somewhat with increasing pHo. Additional experiments showed no significant effect of pHoon holding current or on voltage-activated outward currents (pCai7 with 11 mM EGTA). Our results provide additional evidence for participation of Ca2+channels in regulating basal tone in cerebral smooth muscle and indicate that pHoregulates current through slowly inactivating, dihydropyridine-sensitive L-type Ca2+channels.