The classical type of transient receptor potential channel (TRPC) is a molecular candidate Ca²⁺-permeable cation channel in mammalian cells. TRPC5 is rapidly desensitized after activation by G protein coupled receptor. Here we investigated the desensitization of mTRPC5 and localized the molecular determinants of this desensitization by mutagenesis. TRPC5 was initially activated by muscarinic stimulation using 100 µM carbachol and then decayed rapidly even in the presence of carbachol (desensitization). Increased EGTA or omitting MgATP in the pipette solution slowed the rate of this desensitization. The protein kinase C (PKC) inhibitors, 1 µM chelerythrine, 100 nM GF109203X, or PKC peptide inhibitor (19-36), inhibited this desensitization of TRPC5 activated by 100 µM carbachol. When TRPC5 current was activated by intracellular GTPS, PKC inhibitors prevented TRPC5 desensitization, and the mutation of TRPC5 T972 to alanine slowed the desensitization process dramatically. We conclude that the desensitization of TRPC5 occurs via PKC phosphorylation, and suggest that threonine at 972 residue of mouse TRPC5 might be required for its phosphorylation by PKC.