Background Our previous study has demonstrated that increased intracoronary perfusion leads to a flow-dependent inversion of T waves on body surface ECG. However, whether increased coronary flow influences ventricular action potential duration measured directly from myocardium is unknown. Methods In six pentobarbital-anesthetized sheep, fresh arterial blood was injected into the left circumflex coronary (LCX) artery at a rate of 6 and 10 mL/min, respectively, in the presence of normal coronary flow. Activation'recovery interval (ARI), an estimate of ventricular action potential duration, was measured from epicardial ECGs acquired from the LCX territory. Results The intracoronary injection prolonged ARI by an average of 21 ± 9 and 33 ± 14 msec, respectively. After pre-treatment with nitro-l-arginine, a nitric oxide synthase inhibitor, intracoronary injection at the rate of 6 and 10 mL/min lead to an ARI increase of 3 ± 2 msec (p >0.05) and 11 ± 6 msec (p <0.05) respectively. Conclusions An increase in coronary flow prolongs ventricular action potential duration in the intact sheep heart. Nitric oxide mediates the injection-induced increase in action potential duration.