Extracellular ATP plays an important role in ischemic preconditioning (IPC) through the activation of P_2y purinoceptors. This study examined whether ATP-stimulated P_2y purinoceptors are coupled to pertussis toxin (PTX)-insensitive G protein and whether activation of this pathway enhances myocardial protection afforded by IPC. The rat was treated with PTX for 48 h, and the heart was then isolated and buffer perfused. The heart underwent IPC by three cycles of 5-min ischemia and 5-min reperfusion before 25 min of global ischemia. Isovolumic left ventricular function was measured, and functional recovery at 30 min after reperfusion was taken as an end point of myocardial protection. PTX pretreatment partially inhibited functional protection by IPC. Treatment with 100 µM 8-(p-sulfophenyl) theophylline (SPT) during IPC had no further effect on PTX-induced inhibition of functional protection by IPC, whereas suramin (300 µM) or reactive blue (RB) (10 µM) completely abolished myocardial protection in the preconditioned heart pretreated with PTX. Supplementation with adenosine (30 µM), ATP (30 µM), or UTP (50 µM) significantly enhanced IPC-induced functional protection, although preconditioning with these nucleotides without IPC had no protective effect. Adenosine-enhanced IPC was inhibited by pretreatment with PTX and SPT but not by suramin or RB, whereas ATP-enhanced IPC was inhibited by suramin or RB in combination with PTX pretreatment. On the other hand, UTP-enhanced IPC was not affected by PTX pretreatment but was inhibited by suramin or RB. Adenosine supplemented IPC without PTX pretreatment and ATP supplemented IPC with PTX pretreatment were not affected by nitric oxide synthase inhibitor N-nitro-L-arginine methyl ester (100 µM). Although the protein kinase C inhibitor Ro318425 (0.3 µM) or tyrosine kinase inhibitor genistein (50 µM) had no significant effect on the functional protection afforded by adenosine-supplemented IPC without PTX pretreatment and ATP-supplemented IPC with PTX pretreatment, the combination of Ro318425 and genistein attenuated functional protection afforded by both the purinoceptor agonist-supplemented IPC. These results suggest the crucial involvement of PTX-sensitive and -insensitive G protein coupled purinoceptors in enhanced IPC by supplementation with adenosine, ATP, and UTP.