The classical type of transient receptor potential channel (TRPC) is a molecular candidate for Ca²⁺-permeable cation channels in mammalian cells. Since TRPC channels have calmodulin (CaM) binding sites at their C-termini, we investigated the effect of CaM on mTRPC5. TRPC5 was initially activated by muscarinic stimulation with 50 µM carbachol and then decayed rapidly even in the presence of carbachol. Intracellular CaM (150 µg/ml) increased the amplitude of mTRPC5 current activated by muscarinic stimulation. CaM antagonists (W-7 and calmidazolium) inhibited mTRPC5 currents when they were applied during the activation of mTRPC5. Pretreatment of W-7 and calmidazolium also inhibited the activation of mTRPC5 current. Inhibitors of myosin light chain kinase (MLCK) inhibited the activation of mTRPC5 currents, whereas inhibitors of CaM-dependent protein kinase II did not. SiRNA against cardiac type MLCK also inhibited the activation of mTRPC5 currents. However, inhibitors of CaM or MLCK did not show any effect on GTPS-induced currents. Application of both Rho kinase inhibitor and MLCK inhibitor inhibited GTPS-induced currents. We conclude that CaM and MLCK modulates the activation process of mTRPC5.