Opioids have been previously shown to confer short-term cardioprotection against a prolonged ischemic insult. Therefore, the present study was designed to determine whether opioids can induce a delayed or “second window” of cardioprotection and to assess the potential involvement of the mitochondrial KATP channel. All rats were subjected to 30 minutes of ischemia and 2 hours of reperfusion (I/R). Control animals, injected with saline 24 hours before I/R, elicited an infarct size/area at risk (IS/AAR) of 62.9+/-3.4. TAN-67, a delta1-opioid receptor agonist, was administered 10 or 30 mg/kg IP 12, 24, 48, or 72 hours before I/R. TAN-67 (10 mg/kg) 12- or 24-hour pretreatment did not significantly reduce IS/AAR (62.1+/-6.3 and 43.3+/-7.3, respectively). Similarly, 12-hour pretreatment with TAN-67 (30 mg/kg) did not reduce IS/AAR (60.0+/-5.6); however, 24-hour pretreatment significantly reduced IS/AAR (34.5+/-5.9). Forty-eight-hour pretreatment with TAN-67 maximally reduced IS/AAR (29.2+/-7.0), and opioid-induced cardioprotection was lost after 72-hour pretreatment (61.7+/-3.8). TAN-67-induced cardioprotection could be abolished by pretreatment with the selective delta1-opioid receptor antagonist 7-benzylidenenaltrexone, BNTX, administered either 30 minutes before TAN-67 given 48 hours before I/R or 10 minutes before I/R in rats previously treated for 48 hours with TAN-67 (59.6+/-3.1 and 58.7+/-3.5, respectively). The involvement of the KATP channel was investigated with 2 inhibitors: glibenclamide, a nonselective KATP channel inhibitor, and 5-hydroxydecanoic acid, selective for the mitochondrial KATP channel in rabbits. Glibenclamide, administered 30 minutes before I/R in 48-hour TAN-67-pretreated rats, completely abolished cardioprotection (60. 4+/-3.2). Similarly, 5-hydroxydecanoic acid, administered 5 minutes before I/R in rats pretreated 48 hours previously with TAN-67, completely abolished cardioprotection (57.8+/-2.5). These results suggest that delta1-opioid receptor stimulation, 24 to 48 hours before an ischemic insult, produces a delayed cardioprotective effect that is possibly the result of mitochondrial KATP channel activation.