Objectives: Conditions for ex vivo gene transfer to the transplanted heart were studied in a model of syngeneic abdominal heterotopic heart transplantation in the rat. Various methods of adenoviral-mediated gene transfer to the transplanted heart were compared. Methods: In the first experiment, a dose response study, an adenoviral vector encoding the β-galactosidase gene was infused into the donor heart with the pulmonary artery open and flushed out prior to performing the transplant. In the second experiment, the effects of clamping the pulmonary artery during vector infusion and not flushing out the viral solution, resulting in vector dwell during the warm ischemia, were examined. Results: In the first experiment, gene transfer was relatively inefficient; however, transgene expression improved with increases in the vector dose (range, 1×107-1×109). The efficiency of gene transfer was significantly greater when the conditions of the second experiment were applied. In all models studied, cardiomyocytes and not vascular endothelial cells were the predominant cell type transduced. Conclusions: This study indicates that the conditions of adenoviral vector delivery are critical for optimizing gene transfer in the transplant setting. In addition, intravascular administration of adenoviral vector to the donor heart results predominantly in cardiomyocyte transgene expression. Copyright © 2001 Elsevier Science B.V.