We administered fructose-1,6-bisphosphate (FDP), 1 mM, to isolated and perfused rabbit hearts submitted, after 90 minutes of equilibration, to an ischemic period (60 minutes at a coronary flow of 0.17 ml/min/g), followed by a period of reperfusion (30 minutes at a coronary flow of 3.6 ml/min/g). FDP was delivered at different times following the experimental protocol: 60 minutes before ischemia and for the entire experiment; 60 minutes before and during ischemia, but not at reperfusion; at the onset of ischemia and during reperfusion; and only during reperfusion. The FDP cardioprotective effect was evaluated in terms of recovery of left ventricular pressure developed during reperfusion, creatine phosphokinase (CPK) and noradrenaline release, mitochondrial function (expressed as yield, RCI, QO2, ADP/O), ATP and creatine phosphate (CP) tissue contents, calcium homeostasis, and by measuring oxidative stress in terms of reduced and oxidized glutathione release and tissue contents. Our data show that the cytoprotective action of FDP is closely related to the time of administration. Optimal myocardial preservation was achieved when it was present prior to ischemia and during reperfusion. When given at the time of ischemia or only on reperfusion, FDP does not exert cardioprotection. The data suggest that the FDP cardioprotective effect is related to improvement of energy metabolism.