Gene therapy for common myocardial diseases will require effective and homogeneous gene delivery throughout the intact heart. We created two experimental models to identify and optimize parameters important for adenovirus-mediated cardiac gene transfer. In cultured rabbit ventricular myocytes, the percentage of infected cells increased with higher absolute numbers of virus particles, longer durations of virus exposure, physiological temperatures, and specific culture media compositions. Simulating the in vitro conditions, we delivered adenovirus to intact rabbit hearts by intracoronary perfusion. The percentage of infected cells increased with higher coronary flow rates, longer virus exposure times, and higher virus concentrations. Under optimal conditions, nearly 100% of myocytes expressed the reporter gene β-galactosidase after ex vivo infection. This novel delivery method, the first to demonstrate virtually complete transduction of any intact organ, could be adapted to achieve widespread gene transfer in vivo.