We performed indirect intracoronary delivery of adenovirus vectors in mice and explored techniques including hypothermia and pharmacological means to increase cardiac gene transfer. Mice were maintained normothermic or cooled to 25 °C. The aorta or both pulmonary artery and aorta were clamped while a needle was advanced into the left ventricular cavity to deliver adenovirus vectors encoding enhanced green fluorescent protein (EGFP) or murine adenylyl cyclase Type VI (AC_VI) with saline, nitroprusside, acetylcholine or serotonin. Clamping was maintained for 30 seconds (normothermia), or 2 minutes (25 °C) after adenovirus. Mice were killed 7 or 21 days later and hearts examined for EGFP expression. Gene transfer was increased, compared to clamping the aorta alone and no cooling: 1) 1.3 fold by hypothermia to extend dwell time; 2) 4.5 fold clamping the aorta and pulmonary artery; 3) 11.4 fold by nitroprusside; 4) 11.8 fold by serotonin and 5) 14.3 fold by acetylcholine. Gene expression remained substantial at 21 days and no significant inflammatory response was seen. Efficacy of the method was tested by performing gene transfer of adenovirus encoding AC_VI. Fourteen days after gene transfer, hearts isolated from mice that received adenovirus encoding AC_VI showed increased contractile function. Indirect intracoronary delivery of adenovirus vectors in mice is associated with efficient cardiac gene transfer and with increased left ventricular function after AC_VI gene transfer.