Extracellular calmodulin (CaM) plays significant roles in many physiological processes, but little is known about its mechanism of regulating stomatal movements. In this paper, whether CaM exists in the guard cell walls ofArabidopsis and whether depolymerization of actin cytoskeleton is involved in extracellular CaM-induced stomatal closing are investigated. It is found that CaM exists in guard cell walls ofArabidopsis, and its molecular weight is about 17 kD. Bioassay using CaM antagonists W7-agarose and anti-CaM serum shows that the endogenous extracellular CaM promotes stomatal closure and delays stomatal opening. The long radial actin filaments in guard cells undergo disruption in a time-dependent manner during exogenous CaM-induced stomatal closing. Pharmacological experiments show that depolymerization of actin cytoskeleton enhances the effect of exogenous CaM-induced stomatal closing and polymerization reduces the effect. We also find that exogenous CaM triggers an increase in [Ca2+]cyt of guard cells. If [Ca2+]cyt increase is blocked with EGTA, exogenous CaM-induced stomatal closure is inhibited. These results indicate that extracellular CaM causes elevation of [Ca2+]cyt in guard cells, subsequently resulting in disruption of actin filaments and finally leading to guard cells closure.