Cardiac gene transfer may serve as a novel therapeutic approach in the treatment of heart disease. For it to reach its full potential, methods for highly efficient cardiac gene transfer must be available to investigators so that informative preclinical data can be collected and evaluated. We have recently optimized AAV-mediated cardiac gene transfer protocols in both the mouse and rat. In the mouse, we have developed a procedure for intrapericardial delivery of vector in the neonate and successfully applied intravenous injections in adult animals. In the rat, we have developed a procedure for direct injection of vector into the myocardium in adults and established a protocol for vector delivery into the left ventricular anterior wall by ultrasound-targeted destruction of microbubbles loaded with AAV. Each protocol can be used to achieve safe and efficient cardiac gene transfer in the model of choice.