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 the whole-cell K_ATP channel current evoked by a reduction of the intracellular ATP concentration to 0.5 mM and exposure to 30 µM pinacidil by 90.5 ± 5.0%. Both 1 mM ADP and AMP also decreased the current by 42.8 ± 9.3% and 9.4 ± 4.8%, respectively, but adenosine did not even at 10 mM. The ATP-induced channel inhibition was hardly observed in the presence of 0.2 mM suramin, 0.2 mM GDPßS, or 0.1 mM compound 48/80, whereas it was not influenced by the presence of 0.1 µM staurosporine or 10 mM BAPTA in the pipette. In the presence of 10 µM wortmannin or absence of ATP at the cytosol, the ATP-induced channel inhibition was irreversible. PIP₂ at 0.1 mM in the outside-out patch pipette prevented ATP-induced channel inhibition. The half-maximal inhibition of channel activity for internal ATP concentrations determined in inside-out membrane patches was 13.8 µM in the presence and 1.12 mM in the absence of 0.1 mM ATP at the external site. 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 in associated with a reduction of the sarcolemmal PIP₂ concentration via stimulation of phospholipase C.