We used patch-clamp techniques to elucidate the regulatory mechanisms of ATP-sensitive K⁺ (K_ATP) channels by stimulation of P₂ purinoceptors in guinea pig ventricular myocytes. Extracellular ATP at 0.1 mM transiently inhibited by 90.5 ± 5.0% the whole cell K_ATP channel current evoked by a reduction in intracellular ATP concentration to 0.5 mM and exposure to 30 µM pinacidil. ADP and AMP (both 1 mM) also decreased the current by 42.8 ± 9.3% and 9.4 ± 4.8%, respectively, but adenosine did not, even at 10 mM. ATP-induced channel inhibition was hardly observed in the presence of 0.2 mM suramin, 0.2 mM guanosine 5'-O-(2-thiodiphosphate), or 0.1 mM compound 48/80, whereas it was not influenced by the presence of 0.1 µM staurosporine or 10 mM 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid in the pipette. In the presence of 10 µM wortmannin or the absence of ATP in the cytosol, the ATP-induced channel inhibition was irreversible. Phosphatidylinositol 4,5-bisphosphate (PIP₂) at 0.1 mM in the outside-out patch pipette prevented ATP-induced channel inhibition. The half-maximal internal ATP concentrations for inhibition of channel activity determined in inside-out membrane patches were 13.8 µM in the presence and 1.12 mM in the absence of 0.1 mM ATP at the external side. It is concluded that activity of K_ATP channels is modulated by extracellular ATP by a mechanism involving P_2Y purinoceptors coupled to GTP-binding proteins associated with reduction of the sarcolemmal PIP₂ concentration via stimulation of phospholipase C.