Opioids confer biphasic (early and late) cardioprotection against myocardial infarction by opening mitochondrial K_ATP channels. It is unknown whether cyclooxygenase-2 (COX-2), which mediates ischemia-induced late preconditioning, also mediates opioidinduced cardioprotection. Isolated perfused rat hearts were subjected to 20 min of global ischemia followed by 20 min of reperfusion. BW373U86 (BW), a -opioid receptor agonist, was administered 1, 12, or 24 h before sacrifice. The recovery of left ventricular developed pressure (LVDP) after ischemia/reperfusion improved when BW was administered 1 or 24 h before ischemia (control: 57±8, BW 1 h: 75±5, BW 24 h: 85±6%) but not when it was administered 12 h before (60±5%). The levels of 6-keto-PGF₁ (a stable metabolite of PGI₂) in coronary effluent after 20 min of reperfusion were higher with 24-h BW pretreatment than in controls (1053±92 vs. 724±81 pg/mL), whereas 6-keto- PGF₁ levels at baseline did not differ. Administration of a selective COX-2 inhibitor, NS-398, abolished the late phase of cardioprotection (recovery of LVDP, 53±8%) and attenuated the increase in PGI₂ (706±138 pg/mL) but did not block the early phase of cardioprotection. The selective COX-1 inhibitor, SC-560, did not affect either phase of protection. Western immunoblotting revealed upregulation of PGI₂ synthase protein 24 h after BW administration without changes in COX-1 and COX-2 protein levels. In conclusion, the late (but not the early) phase of -opioid receptor-induced preconditioning is mediated by COX-2. A functional coupling between COX-2 and upregulated PGI₂ synthase appears to underlie this cardioprotective phenomenon in the rat.