The aim of this study is to determine the signal transduction of membrane stretch on intermediate-conductance Ca²⁺-activated K⁺ (IKca) channels in rat aorta smooth muscle cells using the patch-clamp technique. To stretch the cell membrane, both suction to the rear end of patch pipette and hypotonic shock were used. In cell-attached and inside-out patch configurations, the open probability of IKca channels increased when 20- to 45-mmHg suction was applied. Hyposmotic swelling efficiently increased IKca channel current. When the Ca²⁺-free solution was superfused, the activation of IKca current by the hyposmotic swelling was reduced. Furthermore, gadolinium (Gd³⁺) attenuated the activation of IKca channels induced by hyposmotic swelling, whereas nicardipine did not. In the experiments with Ca²⁺-free bath solution, pretreatment with GF109203X, a protein kinase C (PKC) inhibitor, completely abolished the stretch-induced activation of IKca currents. The stretch-induced activation of IKca channels was strongly inhibited by cytochalasin D, indicating a role for the F-actin in modulation of IKca channels by changes in cell stretching. These data suggest that cell membrane stretch activates IKca channels. In addition, the activation is associated with extracellular Ca²⁺ influx through stretch-activated nonselective cation channels, and is also modulated by the F-actin cytoskeleton and the activation of PKC.