BACKGROUND: Preservation of donor hearts for transplantation has traditionally been performed with the use of static cold storage. We have developed and tested a novel gravity-powered system of cold crystalloid perfusion for prolonged donor heart preservation. METHODS: Greyhounds were anesthetized; their hearts were arrested with cold cardioplegic solution and excised. Hearts were allocated to 12 hours of perfusion preservation (n = 6) or cold storage in ice (n = 5). Non-preserved hearts (n = 5) served as a normal reference group. Perfusion hearts were perfused (20 mL/min, 8-12°C) with a novel oxygenated nutrient-containing preservation solution. After preservation, the recovery of the hearts was assessed in a blood-perfused working heart rig over 2 hours in terms of function, blood lactate level, myocardial adenosine triphosphate, and histology. RESULTS: After 2 hours of reperfusion, in comparison with cold storage hearts, perfused heart function curves showed superior recovery of cardiac output (P = .001), power (P = .001), and efficiency (0.046 ± 0.01 vs 0.004 ± 0.003 joules/mL O2, P = .034). Myocardial adenosine triphosphate content (mmol/mg protein) was reduced significantly from the normal level of 26.5 (15.9, 55.8) to 5.08 (0.50, 10.4) (P = .049) in cold storage hearts but not in perfused hearts. Over a period of 2 hours, lactate levels in the blood perfusate were significantly lower in the perfusion group than in the cold storage group (P < .05). CONCLUSIONS: Continuous hypothermic crystalloid perfusion provides myocardial preservation superior to cold storage for long-term heart preservation, with potential applicability to marginal and donation after circulatory death hearts.