Acute administration of magnesium orotate (Mg-Or) at reperfusion has been previously shown to elicit significant protection in isolated rat hearts. Since recovery of mitochondrial function is mandatory for cardioprotection, the present study was aimed at characterising the effects of Mg-Or on mitochondrial respiration. Isolated male adult rat hearts (n=6/group) subjected to 30 min global ischaemia and 120 min reperfusion were randomised to receive: (i) no additional intervention (Ctrl); (ii) Mg-Or at 28 min of ischaemia (Mg-Or-28I) and (iii) Mg-Or at 3 min of reperfusion (Mg-Or-3R). Mitochondria were isolated at 15 min of reperfusion and oxygen consumption was measured at 37°C by polarographic oxymetry in the presence of NAD and FAD-linked substrates, respectively. Basal (state 2) and ADP-stimulated (state 3) respiratory rates were recorded and expressed as nanoatoms oxygen/min/mg mitochondrial protein and respiratory control ratio (RCR) was calculated. In mitochondria respiring on glutamate/malate a statistically significant increase in state 3 respiratory rates was observed in the Mg-Or-28I group but not in Mg-Or-3R when compared to the ischaemic Ctrl (514±29 and 386±17 vs 350±13, respectively, p<0.001). Subsequently, RCR increased from 6.2±0.38 in Ctrl group to 8±0.28 in Mg-Or-28I group (p<0.05). In the presence of complex II-dependent substrate (succinate+amytal) both administration protocols elicited an important increase in state 3 respiration (Mg-Or-28I: 488±25, Mg-Or-3R: 463±20 vs Ctrl: 355±15, respectively, p<0.001). In isolated rat hearts, magnesium 355+orotate elicited a protocol-dependent improvement of mitochondrial respiration at reperfusion that may contribute to its cardioprotective effect against reperfusion injury.