Objectives Impaired tissue reperfusion may be an important limiting factor for long-term hypothermic cardiac preservation. Cardiac function and recovery of coronary responses to endothelium independent and dependent vasodilators were tested before and after one day of preservation by cold perfusion with a normal extracellular solution containing the contraction uncoupler 2,3, butanedione monoxime (BDM) with or without reperfusion with nitroprusside (NP) plus adenosine (Ade) or plus the nucleoside transport inhibitor, nitrobenzylthioinosine (NBTI).Methods Guinea pig hearts (n = 70) were isolated and perfused at constant pressure during normothermic conditions (37°C). Groups 1 and 2 were perfused for 8 hrs without cold perfusion (time control); group 2 was also infused with arginine vasopressin (AVP) for 6 hrs; groups 3–5 were infused with BDM before, during, and initially after 22 hrs of constant, low flow hypothermic (3.8°C) perfusion; group 3 was also infused with NP and Ade, and group 4 with NP and NBTI, for 10 min before rewarming, during rewarming, and for 20 min during the initial normothermic reperfusion period; group 6 was cold perfused without treatment (cold control). Functional indices and transient effects of 200μM bolus Ade (maximal vasodilation), and infusions of 1μM acetylcholine (Ach, endothelial dependent function), and 100μM NP (endothelial independent function) were compared before and after cold preservation.Results One hr after warm reperfusion, i.e., after 25 hrs, isovolumetric left ventricular pressure (LVP as a % of initial 100% control) was equivalent and greater in groups 3 (77±3% (SEM) and 4 (73±3%) than in groups 5 (53±6%) and 6 (2.3±5%); LVP was similar in groups 1 (85±3%) and 2 (81±2%) after 5 hrs. Before cold perfusion coronary flow (CF) increased similarly in all 6 groups with Ade (200±3%), Ach (120±3%) and NP (130±3%); 1 hr after warm reperfusion baseline CF was greater in groups 3 (81±3%) and 4 (84±2%) than in groups 5 (68±3%) and 6 (53±3%); CF was higher in group 1 (104±2%) than in group 2 (72±1%). CF responses after warm reperfusion in groups 1–6, respectively, were: 176±4, 130±2, 115±3, 109±2, 93±3, and 80±5% for Ade; 123±f 5, 105±2, 69±3, 73±3, 70±3, and 57±3% for Ach; and 123±4, 111±2, 86±3, 88±4, 91±4, and 73±4% for NP.Conclusions After long-term hypothermic perfusion with normal extracellular solution, flow responses to endothelium dependent, independent and mixed vasodilators are severely blunted. In part, this may be due to reduced baseline flow following normothertnic reperfusion because infusion of BDM during hypothermia, with infusion of NP plus Ade or NBTI during normothermic reperfusion, improves CF responses to both endothelium dependent and independent vasodilators.