An increase in cytosolic free calcium has been shown to occur during ischemia in perfused hearts and plays a pivotal role in ischemia/reperfusion injury. The objective of this study was to investigate the contributions of Na(+)/H(+) exchange and Na(+)/Ca(2+) exchange to changes in intracellular calcium ([Ca(2+)](i)) during simulated ischemia and reperfusion in quiescent isolated rat cardiac myocytes. [Ca(2+)](i) was measured by laser confocal microscope using the fluorescent indicator Fluo 3 and expressed as the corrected intensity of Fluo 3 fluorescence. [Ca(2+)](i) increased to 140.3+/-13.0% (P<0.05 vs preischemic control 100%) after 5 min simulated ischemia, and remained at high level of 142.8+/-15.5% (P<0.05) after the following 15 min reperfusion. The increase in [Ca(2+)](i) during simulated ischemia and reperfusion was suppressed by 100 micromol/L amiloride (inhibitor of Na(+)/H(+) exchanger), 5 mmol/L NiCl2 (inhibitor of Na(+)/Ca(2+) exchanger) and calcium-free solution; [Ca(2+)](i) was 101.4+/-16.3%, 110.4+/-11.1% and 107.1+/-10.8%, respectively, after 5 min simulated ischemia, and 97.8+/-14.3%, 106.2+/-14.5% and 106.6+/-15.7%, respectively, after 15 min reperfusion. Compared with control cells, the amplitude of spontaneous calcium oscillation was lessened in cells treated with Ca-free perfusion and NiCl2 during reperfusion. In addition, no calcium oscillation was observed in cells pretreated with amiloride. These results suggest that Na(+)/H(+) exchange and Na(+)/Ca(2+) exchange are activated during simulated ischemia in isolated quiescent cardiac myocytes, leading to the elevation of [Ca(2+)](i) induced by simulated ischemia and reperfusion.