Background: Hypothermic conditions required for donor heart preservation may reduce gene-transfer efficiency. Experiments were designed to determine whether a perfusion technique could improve the efficiency of gene transfer to donor hearts. Methods: An adenoviral vector encoding β-galactosidase (3.5 x 108 plaque-forming units) was infused into explanted rat hearts under 4 conditions (each n = 6): (1) the virus was diluted in 350 μL of University of Wisconsin solution and infused as a high-pressure bolus into the coronary arteries of donor hearts through the aortic root; (2) the virus was diluted in 5 mL of University of Wisconsin solution and circulated by means of a peristaltic pump (flow, 0.75 mL/min) through the vasculature of the donor heart for 30 minutes; (3) 5 mL of viral solution was circulated as for group 2 for 15 minutes; and (4) 5 mL of viral solution was circulated for 5 minutes at a flow rate of 2.4 mL/min. Transduced hearts were transplanted into the abdomen of syngeneic rats, and transgene expression was assessed by means of immunoassay 4 days later. Results: The median β-galactosidase content was (1) 45.0 ng/mg protein (25th-75th percentile, 33-73 ng/mg), (2) 640 ng/mg protein (25th-75th percentile, 614-878 ng/mg), (3) 493.8 ng/mg protein (25th- 75th percentile, 456-527 ng/mg), and (4) 503.3 ng/mg protein (25th-75th percentile, 475-562 ng/mg; P < .01 for group 2 vs group 1, and P < .05 for groups 3 and 4 vs group 1). Transgene expression was predominantly in myocytes and favored the subepicardial region of the right ventricle. Conclusion: Hypothermic perfusion of the donor heart with an adenoviral vector resulted in efficient transgene expression compared with that induced by a single bolus injection.