BACKGROUND The beneficial effect of ischemic preconditioning (PC) has been extensively studied in normal hearts but its effects on diseased hearts remain largely unknown. The effect of PC in the already ischemic myocardium has not been previously studied, although ischemia in varying intervals, which is difficult to assess, is often encountered in clinical practice. OBJECTIVE To investigate whether the cardioprotective effect of PC is preserved when it is applied after a period of ischemia of varying duration. METHODS Male Wistar rats were used for this study. Isolated normal rat hearts were perfused in Langendorff mode. Before 20 min of zero flow global ischemia followed by 45 min of reperfusion, hearts were subjected to an initial 20-min period of ischemia followed by 10 min of reperfusion (group A1); an initial 20-min period of ischemia followed by 10 min of reperfusion and two-cycle PC (3 min of ischemia, 5 min of reperfusion followed by 5 min of ischemia and 5 min of reperfusion) (group A2); and two-cycle PC followed by the initial 20-min period of ischemia and 10 min of reperfusion (group A3). Groups B and C were subjected to an initial ischemia of 15 min and 10 min, respectively, and subgroups 1, 2 and 3 were treated as above. Left ventricular end-diastolic pressure was measured at 45 min of reperfusion (LVEDP45 in mmHg). Postischemic recovery of left ventricular developed pressure was expressed as a percentage of the initial value (LVDP%). RESULTS LVDP% and LVEDP45 were similar between groups A1 and A2, while when ischemic preconditioning preceded the two periods of ischemia (group A3), it resulted in significantly higher LVDP% and significantly lower LVEDP45 compared with groups A1 and A2. Left ventricular functional recovery was not increased in group B2 compared with group B1. LVDP% and LVEDP45 were similar among groups C1, C2 and C3. CONCLUSIONS Ischemic preconditioning does not improve functional recovery in isolated rat hearts that have been initially subjected to 20 min or 15 min of zero-flow global ischemia, while an initial 10-min ischemic period seems to precondition the heart.