Objective: Nitric oxide (NO) is reported to be both protective and detrimental in models of myocardial ischaemia/reperfusion injury, which may be concentration dependent. Our objective was to characterise this dichotomy using the nitric oxide donor, S-nitroso N-acetyl penicillamine (SNAP) in isolated perfused mouse heart and isolated mouse cardiac mitochondria. Methods: To determine the effect of nitric oxide concentration on myocardial viability, isolated mouse hearts were subjected to 35 min global ischaemia and 30 min reperfusion in the presence of SNAP (0.02–20 μM). To determine whether NO mediated protection was via opening of the putative mitochondrial KATP channel and/or free radical synthesis, SNAP perfused hearts were also treated with the mitochondrial KATP channel blocker, 5-hydroxy decanoate (5-HD) and the free-radical scavenger, N-(2-mercaptopropionyl)-glycine (MPG). This data was correlated with mitochondrial membrane potential (ΔΨm), measured with the potentiometric dye, tetra-methyl rhodium methyl ester (TMRM), in isolated mitochondria,by flow cytometry. Results: SNAP dose-dependently attenuated infarct size, with maximal protection observed at 2 μM (17±4% versus controls 32±3%, P<0.01). At greater concentrations however, protection was lost with infarct sizes tending towards control at 20 μM (29±3%). These results were paralleled by changes in ΔΨm in the isolated mitochondria: ΔΨm depolarisation peaking with 1 μM SNAP (26±4% shift in TMRM fluorescence, P<0.01); at greater concentrations, this relationship was lost. The mitochondrial KATP channel blocker, 5-HD, resulted in both abrogation of SNAP infarct size reduction and concomitant loss of ΔΨm depolarisation in the mitochondria. MPG however did not influence the cardioprotective properties of SNAP. Conclusion: We demonstrate that nitric oxide can mediate cardioprotection in a dose-dependent fashion by an effect that may be related to ΔΨm. Both cardioprotection and ΔΨm changes are sensitive to 5-HD and the cardioprotection appears independent of free-radical synthesis.