In the COS7 cells transfected with cDNAs of the Kir6.2, SUR2A, and M₁ muscarinic receptors, we activated the ATP-sensitive potassium (K_ATP) channel with a K⁺ channel opener and recorded the whole-cell K_ATP current. The K_ATP current was reversibly inhibited by the stimulation of the M₁ receptor, which is linked to phospholipase C (PLC) by the G_q protein. The receptor-mediated inhibition was observed even when protein kinase C (PKC) was inhibited by H-7 or by chelating intracellular Ca²⁺ with 10 mM 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetate (BAPTA) included in the pipette solution. However, the receptor-mediated inhibition was blocked by U-73122, a PLC inhibitor. M₁-receptor stimulation failed to inhibit the K_ATP current activated by the injection of exogenous phosphatidylinositol 4,5-bisphosphate (PIP₂) through the whole-cell patch pipette. The receptor-mediated inhibition became irreversible when the replenishment of PIP₂ was blocked by wortmannin (an inhibitor of phosphatidylinositol kinases), or by including adenosine 5′-[β,γ–imido]triphosphate (AMPPNP, a nonhydrolyzable ATP analogue) in the pipette solution. In inside-out patch experiments, the ATP sensitivity of the K_ATP channel was significantly higher when the M₁ receptor in the patch membrane was stimulated by acetylcholine. The stimulatory effect of pinacidil was also attenuated under this condition. We postulate that stimulation of PLC-linked receptors inhibited the K_ATP channel by increasing the ATP sensitivity, not through PKC activation, but most probably through changing PIP₂ levels.