The mechanism of flavonol-induced cardioprotection is unclear. We compared the protective actions of a flavonol that inhibits calcium utilization and has antioxidant activity, 3',4'-dihydroxyflavonol (DiOHF); a flavonol that affects only calcium activity, 4'-OH-3'-OCH(3)-flavonol (4'-OH-3'-OCH(3)F); and a water-soluble flavonol with selective antioxidant activity, DiOHF-6-succinamic acid (DiOHF-6-SA), in isolated, perfused rat hearts. Hearts were subjected to global ischemia for 20 min followed by 30 min reperfusion and were treated with vehicle (0.05% DMSO), DiOHF, 4'-OH-3'-OCH(3)F, or DiOHF-6-SA (all 10 μM, n=5-8 per group). Flavonols were infused for 10 min before ischemia and during reperfusion. In vehicle-treated hearts, left-ventricular (LV) +dP/dt was reduced by 60% at the end of reperfusion compared to the preischemic level. Lactate dehydrogenase (LDH) release was elevated and endothelial NO synthase (eNOS) expression was lower in vehicle-treated hearts compared to shams. In comparison, DiOHF treatment improved LV function upon reperfusion, decreased LDH, and preserved eNOS expression. The antioxidant DiOHF-6-SA also preserved contractility, reduced LDH, and preserved eNOS expression. In contrast, hearts treated with 4'-OH-3'-OCH(3)F showed a degree of contractile impairment similar to that of the vehicle group. DiOHF and DiOHF-6-SA also exerted cardioprotection when given only during reperfusion and not when administered only before ischemia. Flavonol-induced cardioprotection relies on antioxidant activity and is mainly exerted during reperfusion.