Achieving efficient cardiac gene transfer in a large animal model has proven to be technically challenging. Prior strategies have employed cardio-pulmonary bypass or dual catheterization with the aid of vasodilators to deliver vectors, such as adenovirus, adeno-associated virus (AAV) or plasmid DNA. While single-stranded AAV vectors have shown the greatest promise, they suffer from delayed expression, which might be circumvented by using self-complementary vectors. We have recently optimized a cardiac gene transfer protocol in the canine using a percutaneous transendocardial injection catheter to deliver an AAV vector under fluoroscopic guidance. Percutaneous transendocardial injection of self-complementary AAV (scAAV)-6 is a safe, effective method for achieving efficient cardiac gene transfer to approximately 60% of the myocardium.