Background: To facilitate the application of adenoviral gene therapy in clinical heart transplantation, we developed an ex vivo hypothermic recirculatory adenoviral gene transfer method to the transplanted pig heart. Methods: Experimental animals were assigned into three groups; controls, 1 × 108 plaque-forming units (pfu)/ml group and 1 × 109 pfu/ml group. During the 30 min gene transfer perfusion, 200 ml of University of Wisconsin solution containing the adenoviral vector was recirculated through the coronary vessels. The myocardial temperature was maintained below 4°C and the perfusion pressure was adjusted at 50 mmHg. Results: Cardiac myocyte transduction efficiencies in the 1 × 1 08 pfu/ml group were 0.04% and 0.07%, whereas transduction efficiencies in the 1 × 109 pfu/ml group were widely distributed from 0.45% to 22.62%. The gene transduction efficiency increased with the virus titer. Additionally, no difference in the transduction efficiency was observed between different segments of the left ventricle. The current gene transfer method at 1 × 109 pfu/ml of adenovirus titer enabled homogeneous gene transduction into the transplanted pig heart up to a maximum of 22.62%. Conclusions: This model can be applied to a large isolated heart and will greatly facilitate the investigation of gene therapy in large animal models of heart transplantation. Copyright © 2006 John Wiley & Sons, Ltd.