Mitochondrial permeability transition (MPT) pores have recently been implicated as a potential mediator of myocardial ischemic injury. Nitric oxide (NO) donors induce a powerful late phase of cardioprotection against ischemia-reperfusion injury; however, the cellular mechanisms involved are poorly understood. The role of MPT pores as a target of cardioprotective signaling pathways activated by NO has never been explored in detail. Thus mice were administered the NO donor diethylenetriamine (DETA)/NO (4 doses of 0.1 mg/kg iv each) 24 h before 30 min of coronary artery occlusion followed by 24 h of reperfusion. Infarct size was significantly reduced in DETA/NO-treated mice (30 ± 2% of risk region in treated mice vs. 50 ± 2% in control mice; P < 0.05), which demonstrates powerful cardioprotection. To examine the role of MPT pores, mice were administered atractyloside (Atr; 25 mg/kg iv), which induces adenine nucleotide translocase-dependent MPT, 20 min before ischemia. Atr blocked the infarct-sparing effects of DETA/NO (infarct size, 58 ± 1 vs. 30 ± 2% of risk region in DETA/NO; P < 0.05), whereas Atr alone had no effect. Mitochondria isolated from DETA/NO-treated mice exhibited increased resistance to Ca²⁺-induced swelling by 20 μmol/l CaCl₂ or by the higher concentration of 200 μmol/l, which suggests that cardioprotection involves decreased propensity for MPT. Preincubation of mitochondria from control hearts with 30 nmol/l of the pore inhibitor cyclosporin A prevented swelling by 200 μmol/l CaCl₂, thereby confirming that Ca²⁺ induces mitochondrial swelling via MPT. In accordance with the effects on infarct size, administration of Atr to the mice significantly abrogated DETA/NO-induced protection against Ca²⁺-induced mitochondrial swelling. These phenotypic alterations were associated with an increase in the antiapoptotic protein Bcl-2, which suggests that the underlying mechanisms may involve inhibition of cell death by Bcl-2. These data suggest that a critical process during NO donor-induced cardioprotection is to prevent MPT pore opening potentially via targeting of the adenine nucleotide translocator.