Short episodes of ischemia and reperfusion (ischemic preconditioning) have been shown to attenaute the detrimental rise in intracellular free Ca²⁺ ([Ca²⁺]_i) caused by subsequent sustained ischemia. The purpose of this study was to investigate the detailed time course of [Ca²⁺]_i during such short episodes of ischemia and reperfusion. [Ca²⁺]_i was measured at a high time resolution by surface fluorometry and indo-1 in isolated perfused rat hearts during three episodes of 5 minutes of ischemia followed by 5 minutes of reperfusion. We found that the rise in systolic [Ca²⁺]_i was significantly smaller during the second (191% ± 67%) and third (194% ± 75%) episodes of ischemia than during the first episode (289% ± 75%, P < 0.05 vs both subsequent episodes). This attenuated rise in systolic [Ca²⁺]_i was preserved during perfusion with low extracellular Na⁺ (105.5 mmol/L [Na⁺]_o). During short episodes of ischemia and reperfusion, [Ca²⁺]_i rises less during the second and third ischemic episode than during the first episode. Thus one transient ischemic stimulus led to a rapid adaptation of [Ca²⁺]_i homeostasis to subsequent ischemic conditions. Such a rapid adaptation might be an important mechanism of the ischemic preconditioning phenomenon in protecting against ischemic injury.