The present work investigates the effect of phosphatidylinositol- 4, 5- bisphosphate (PIP2) on native TRPC6 channel activity in freshly dispersed rabbit mesenteric artery myocytes using patch clamp recording and co- immunoprecipitation methods. Inclusion of 100 µM diC8- PIP2 in the patch pipette and bathing solutions respectively inhibited angiotensin II (Ang II)- evoked whole- cell cation currents and TRPC6 channel activity by over 90 %. In inside- out patches diC8- PIP2 also inhibited TRPC6 activity induced by the diacylglycerol analogue 1- oleoyl- 2- acetyl- sn- glycerol (OAG) with an IC50 of 7.6 µM. Anti- PIP2 antibodies potentiated Ang II- and OAG- evoked TRPC6 activity by about 2- fold. Depleters of tissue PIP2 wortmannin and LY294002 stimulated TRPC6 activity, as did the polycation PIP2 scavenger poly- L- lysine. Wortmannin reduced Ang II- evoked TRPC6 activity by over 75 % but increased OAG- induced TRPC6 activity by over 50- fold. Co- immunoprecipitation studies demonstrated association between PIP2 and TRPC6 proteins in tissue lysates. Pretreatment with Ang II, OAG and wortmannin reduced TRPC6 association with PIP2. These results provide for the first time compelling evidence that constitutively produced PIP2 exerts a powerful inhibitory action on native TRPC6 channels.