Ca²⁺ is the main trigger for mitochondrial permeability transition pore opening, which plays a key role in cardiomyocyte death after ischemia reperfusion. We investigated if a reduced accumulation of mitochondrial Ca²⁺ might explain the attenuation of lethal reperfusion injury by postconditioning. Anesthetized New Zealand White rabbits underwent 30 min of ischemia followed by either 240 (infarct size protocol) or 60 (mitochondria protocol) min of reperfusion. They received either no intervention (control), preconditioning by 5 min. ischemia / 5 min. reperfusion, postconditioning by 4 cycles of 1 min. reperfusion / 1 min. ischemia, at the onset of reflow, or pharmacological inhibition of the transition pore opening by NIM811 (5 mg/kg, IV) given at reperfusion. Area at risk and infarct size were assessed by blue dye injection and TTC staining. Mitochondria were isolated from the risk region, for measurement of (1) Ca²⁺ retention capacity (CRC), and (2) mitochondrial content of total (atomic absorption spectrometry) and ionized (potentiometric technique) calcium concentration. CRC averaged 0.73 ± 0.16 in control versus 4.23 ± 0.17 µg Ca²⁺/ mg proteins in shams (p<0.05). Postconditioning, preconditioning or NIM811 significantly increased CRC (p<0.05 vs control). In the control group, total and free mitochondrial calcium significantly increased to 2.39 ± 0.43 and 0.61 ± 0.10, respectively, versus 1.42 ± 0.09 and 0.16 ± 0.01 µg Ca / mg in sham (p<0.05). Surprisingly, whereas total and ionized mitochondrial Ca²⁺ decreased in preconditioning, it significantly increased in postconditioning and NIM811 groups. These data suggest that retention of calcium within mitochondria may explain the decreased reperfusion injury in postconditioned (but not preconditioned) hearts.