Oxidative stress, activation of intracellular protein kinases and cardiomyocyte apoptosis are known mediators of cardiac ischaemia/reperfusion injury. The sites at which NP202, a novel water soluble pro-drug of 3',4'-dihydroxyflavonol (DiOHF), acts in this cascade to cause cardioprotection are unknown. In this study we examined the ability of NP202 to reduce infarct size after a prolonged period of ischaemia and reperfusion. In addition, we tested whether NP202 inhibits pro-apoptotic signalling, apoptosis and inflammation following myocardial ischaemia and reperfusion. Sheep were anaesthetised, the heart exposed and the 2nd branch of the left anterior descending coronary artery isolated. The artery was occluded for 3h and, five minutes before 3h of reperfusion was commenced, sheep were treated with intravenous vehicle or NP202. At the end of reperfusion infarct size was measured and normal left ventricle, non-infarcted area-at-risk and infarcted myocardium were collected to identify polymorphonuclear leukocytes (PMN) or apoptotic cells (TUNEL-positive), or assessed for activation of mitogen-activated protein kinase (MAPK) pathways by Western blot analysis. Compared with vehicle treatment, NP202 reduced infarct size (-20 ± 4%, P<0.05) and decreased the number of PMNs and TUNEL-positive cells in the area-at-risk (-35 ± 16% and -52 ± 19%, respectively) and infarcted tissue (-57 ± 9 and -81 ± 5%, respectively, P<0.05). Furthermore, NP202 significantly reduced I/R-induced elevated p38 MAPK phosphorylation (by 67 ± 4%, P<0.05) in the area-at-risk zone. In conclusion, the novel aqueous flavonol NP202 provided significant cardioprotection from clinically relevant prolonged myocardial ischaemia when administered just before reperfusion. Efficacy of NP202 was also associated with reduced p38 MAPK activation, inflammation and apoptotic cell death.