Background: In this investigation we studied the efficacy and durability of recombinant adeno-associated virus serotype 9 (rAAV9) vector-mediated gene transfer to the transplanted rat heart. Methods: A rAAV9-CMV-lacZ vector diluted in cold (4°C) University of Wisconsin solution was used to perfuse the rat coronary vasculature for 20 minutes prior to syngeneic heterotopic transplantation. Perfusion experiments (six groups, n = 3/group) were performed without rAAV9 and at four separate doses ranging from 2 × 109 to 2 × 1012 viral genomes/ml. The transplanted heart was recovered 10 days or 3 months after transplantation and expression of lacZ assessed by histology, enzyme-linked immunoassay and real-time reverse transcript-polymerase chain reaction (RT-PCR). In a final group (n = 3), rAAV9 was administered systemically to compare the cardiac transduction efficiency and viral distribution to other organs. Results: Transduction efficiency of perfused virus correlated with vector dose (p < 0.0001), with myocardial transduction ranging up to 71.74% at the highest dose. Cardiac expression of lacZ was equivalent at 10 days and 3 months. There was no evidence of viral gene transfer to other organs after heart transplantation. Conclusions: Our findings demonstrate efficient and durable rAAV9-mediated gene transfer to the transplanted heart after ex vivo perfusion and suggest that AAV9 is a promising vector for cardiac gene therapy. © 2008 International Society for Heart and Lung Transplantation.