Objective: To confirm gene transfer techniques especially into the whole heart, we tried out a gene transfer method involving liposome with the viral envelope hemagglutinating virus of Japan liposome as an alternative to existing techniques such as cationic lipofection or other viral vectors. Method: For this study, hemagglutinating virus of Japan liposome (H group) or cationic liposome(L group) was used to compare the efficacy of gene transfection of oligonucleotide labeled with fluorescein isothiocyanate and cDNA of β-galactosidase and human manganese-superoxide dismutase. Fluorescein-labeled oligonucleotide, cDNA of β-galactosidase, or manganese- superoxide dismutase was complexed with liposomes, DNA-binding nuclear protein, and the vital protein coat of hemagglutinating virus of Japan. After donor rat hearts arrested by cardioplegia had been harvested, the coronary artery during cardioplegic arrest was infused via an aortic cannula with the liposome-gene complex. Next, the hearts were transplanted into the abdomen of recipient rats of the same strain, and all recipients were put to death after 3 days of transfection. Results: Fluorescein isothiocyanate was detected in the nuclei of more than 70% of the myocytes (75% ± 14%, n = 5) in the H group compared with fewer than 10% in the L group (7% ± 5%, n = 5). The intensity of fluorescein isothiocyanate was significantly higher in the H group (979 ± 112 FI) than in the L group (116 ± 68 FI). β-Galactosidase was expressed in the cytosol of more than 50% of the myocytes in the H group (61% ± 7%, n = 5) compared with none in the L group (0%, n = 5). After 3 days of gene transfection, and when exposed to ischemia (30 minutes, 37°C) and reperfusion (30 minutes, 37°C) with Langendorff apparatus, the hearts transfected with manganese-superoxide dismutase (S group, n = 5) showed a significantly higher percentage of recovery of left ventricular end-diastolic pressure (S vs C, 86% ± 3% vs 54% ± 12%) and coronary flow (98% ± 2% vs 66% ± 12%) than did the control hearts (C group, n = 5). Western blotting analysis showed an apparent increased expression of manganese-superoxide dismutase in the hearts transfected with manganese-superoxide dismutase compared with the control hearts. These results clearly demonstrated that the donor hearts were transfected with fluorescein-labeled oligonucleotide and the β-galactosidase gene as a result of coronary infusion of the hemagglutinating virus of Japan liposome during cardioplegic arrest at the time of harvest. Furthermore, the hearts transfected with manganese- superoxide dismutase showed significant improvement in tolerance against ischemia reperfusion injury. Conclusion: We believe that this method represents a novel in vivo gene transfer technique for the heart and thus may provide a new tool for research and therapy of heart transplantation.