In the present study, we hypothesized that postcon (postconditioning) confers cardioprotection in vivo by reducing the production of ONOO- (peroxynitrite) and nitro-oxidative stress subsequent to the inhibition of the iNOS (inducible NO synthase). Patients with AMI (acute myocardial infarct) were randomly assigned to undergo percutaneous coronary intervention without (control) or with ischaemic postcon by three episodes of 30-s inflation and 30-s deflation of the angioplasty balloon. Animal models of MI/R (myocardial ischaemia/reperfusion) injury were induced in rats by occluding the left coronary artery for 40 min followed by 4-h reperfusion. Rats were randomized to receive vehicle, postcon (three cycles of 10-s reperfusion and 10-s coronary re-occlusion preceding full reperfusion), the selective iNOS inhibitor 1400W or postcon plus 3-morpholinosydnonimine (an ONOO- donor). Postcon in patients reduced iNOS activity in white blood cells, decreased plasma nitrotyrosine, a fingerprint of ONOO- and an index of nitro-oxidative stress, and improved cardiac function (P<0.01 compared with control). In rats, postcon reduced post-ischaemic myocardial iNOS activity and nitrotyrosine formation, reduced myocardial infarct size (all P<0.05 compared with control) and improved cardiac function. Administration of 1400W resembled, whereas 3-morpholinosydnonimine abolished, the effects of postcon. In conclusion, reduction in ONOO--induced nitro-oxidative stress subsequent to the inhibition of iNOS represents a major mechanism whereby postcon confers cardioprotection in vivo.