Nitric oxide (NO) has the potential to be beneficial in attenuating ischaemia–reperfusion (IR) injury, or deleterious due to its tendency to form reactive nitrogen species (RNS). In addition, different donors release varying amounts of NO, making them difficult to compare directly. We studied the effects of two NO donors, SNAP (S-nitroso-N-acetyl-DL-penicillamine) and DEA/NO (diethylamine NONOate). Isolated rat hearts were perfused with or without SNAP at 2–100 μM or DEA/NO at 2–20 μM. The NO donors were added 20 minutes before the onset of ischaemia (30 minutes) and remained present throughout reperfusion (60 minutes). Injury was assessed by measuring left ventricular developed pressure (LVDP) and lactate dehydrogenase (LDH) release. SNAP significantly protected hearts from IR injury at 20 and 40 μM, but not 2 μM or 100 μM, compared with controls, as indicated by improved recovery of LVDP and reduced LDH release (n=6 for each condition). Protection with DEA/NO was seen at 2 μM but at 20 μM protection was lost. In conclusion, NO donors protected hearts, but the effect was concentration dependent; protection being lost at higher levels of the NO donor, possibly by formation of RNS. In addition, the optimum concentration for cardioprotection was different for each donor.