A hyposmotic challenge elicited contraction of isolated canine basilar arteries. The contractile response was nearly abolished by the removal of extracellular Ca²⁺ and by the voltage-operated Ca²⁺ channel blocker nicardipine, but was unaffected by thapsigargin, which depletes intracellular Ca²⁺ stores. The contraction was also inhibited by Gd³⁺ and ruthenium red, cation channel blockers, and DIDS and niflumic acid, Cl^- channel blockers. The reduction of extracellular Cl^- concentrations enhanced the hypotonically induced contraction. Patch clamp analysis showed that a hyposmotic challenge activated outwardly-rectifying whole-cell currents in isolated canine basilar artery myocytes. The reversal potential of the current was shifted toward negative potentials by reductions in intracellular Cl^- concentrations, indicating that the currents were carried by Cl^-. Moreover, the currents were abolished by 10 mM BAPTA in the pipette solution and by the removal of extracellular Ca²⁺. Taken together, these results suggest that a hyposmotic challenge activates cation channels, which presumably cause Ca²⁺ influx, thereby activating Ca²⁺-activated Cl^- channels. The subsequent membrane depolarization is likely to increase Ca²⁺ influx through voltage-operated Ca²⁺ channels and elicit contraction.