Extracellular ATP is a parent molecule of adenosine, a principal mediator of ischemic preconditioning (IPC), but itself may play an important role in IPC through the activation of P2Y purinoceptors. The present study has further examined the hypothesis that ATP-stimulated P2Y purinoceptors is coupled to pertussis toxin (PTX)-insensitive G protein and activation of this pathway enhances myocardial protection afforded by IPC. The rat was treated with PTX or its vehicle for 48 hours and the heart was isolated and buffer perfused. The heart underwent IPC by 3 cycles of 5 minutes ischemia and 5 min reperfusion before 25 minutes of global ischemia. Isovolumic left ventricular function was measured and functional recovery at 30 minutes after reperfusion was taken as an end-point of myocardial protection. PTX pretreatment partially but significantly inhibited functional protection by IPC. Treatment with 8-(p-sulfophenyl) theophylline (SPT; 100 µM) during IPC had no further effect on PTX-induced inhibition of functional protection by IPC, while 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) during IPC significantly enhanced functional protection over IPC, although time-matched 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, while 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 (NO) synthase inhibitor N-nitro-L-arginine methyl ester (L-NAME; 100 µM). Although protein kinase C inhibitor Ro318425 (0.3 µM) or tyrosine kinase inhibitor genistein (50 µM) each alone had no significant effect on functional protection afforded by adenosine-supplemented IPC without PTX pretreatment and ATP-supplemented IPC with PTX pretreatment, 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 conventional as well as in enhanced IPC by supplementation with adenosine, ATP and UTP in the rat heart. Enhanced IPC afforded by PTX-sensitive and -insensitive G protein-coupled purinoceptors does not require endogenous NO but is triggered by a cooperative interaction of PKC with tyrosine kinase signaling pathways.