Extracellular ATP is known to augment cardiac contractility by increasing the intracellular Ca²⁺ concentration ([Ca²⁺]_i) in cardiomyocytes; however, the status of ATP-mediated Ca²⁺ mobilization in hearts undergoing ischemia-reperfusion (I/R) has not been examined previously. In this study, therefore, isolated rat hearts were subject to 10 to 30 min of global ischemia and 30 min of reperfusion, and the effect of extracellular ATP on [Ca²⁺]_i was measured in purified cardiomyocytes by Fura-2 microfluorometry. Reperfusion for 30 min of 20 min ischemic hearts, unlike 10 min ischemic hearts, revealed a partial depression in cardiac function and ATP-induced increase in [Ca²⁺]_i; no changes in basal [Ca²⁺]_i were evident in 10 or 20 min I/R preparations. On the other hand, reperfusion of 30 min ischemic hearts for 5, 15, or 30 min showed a marked depression in both cardiac function and ATP-induced increase in [Ca²⁺]_i in addition to a dramatic increase in basal [Ca²⁺]_i. The positive inotropic effect of extracellular ATP was attenuated and the maximal binding characteristics of [35S]ATPS with crude membranes from hearts undergoing I/R was decreased. The ATP-induced increase in [Ca²⁺]_i in cardiomyocytes was depressed by verapamil and cibacrone blue in both control and I/R hearts; however, this response in the I/R hearts, unlike the control hearts, was not affected by ryanodine. The I/R-induced alterations in cardiac function and ATP-induced increase in [Ca²⁺]_i were attenuated by treatment with an antioxidant mixture as well as by ischemic preconditioning. The observed changes due to I/R were simulated in hearts perfused with hydrogen peroxide. The results suggest an impairment of extracellular ATP-induced Ca²⁺ mobilization in the I/R hearts and this defect appears to be mediated through oxidative stress.