Using the whole-cell and single channel recording techniques, the influence of actin cytoskeletons on L-type Ca2+ current was investigated in human gastric smooth muscle cells. In isotonic condition, an actin depolymerizer cytochalasin D (Cyt-D) markedly decreased the whole-cell current (I(Ba)) without changing steady-state voltage dependency and single channel conductance. Intracellular dialysis of phalloidin, an actin polymerizer, significantly increased the I(Ba). Hypotonic stretch (222 mOsm/L) of the myocytes increased the I(Ba), and Cyt-D significantly inhibited the I(Ba) increase by the stretch. Phalloidin was without effect on the I(Ba) increase by the stretch. Phalloidin antagonized the Cyt-D inhibition of the stretch-induced I(Ba) increase. Neither heterotrimeric G protein modifiers (GTPgammaS and GDPbetaS) nor rho GTPase inhibitor (C3 exoenzyme) influenced the stretch-induced responses. These results reveal that the integrity of the actin cytoskeleton is an important factor which determines the activity of L-type Ca2+ channels and a response to stretch.