We used a catheter-based technique to achieve generalized cardiac gene transfer in vivo and to alter cardiac function by overexpressing phospholamban (PL) which regulates the activity of the sarcoplasmic reticulum Ca²⁺ ATPase (SERCA2a). By using this approach, rat hearts were transduced in vivo with 5 × 10⁹ pfu of recombinant adenoviral vectors carrying cDNA for either PL, β-galactosidase (β-gal), or modified green fluorescent protein (EGFP). Western blot analysis of ventricles obtained from rats transduced by Ad.PL showed a 2.8-fold increase in PL compared with hearts transduced by Ad.βgal. Two days after infection, rat hearts transduced with Ad.PL had lower peak left ventricular pressure (58.3 ± 12.9 mmHg, n = 8) compared with uninfected hearts (92.5 ± 3.5 mmHg, n = 6) or hearts infected with Ad.βgal (92.6 ± 5.9 mmHg, n = 6). Both peak rate of pressure rise and pressure fall (+3, 210 ± 298 mmHg/s, −2, 117 ± 178 mmHg/s, n = 8) were decreased in hearts overexpressing PL compared with uninfected hearts (+5, 225 ± 136 mmHg/s, −3, 805 ± 97 mmHg/s, n = 6) or hearts infected with Ad.βgal (+5, 108 ± 167 mmHg/s, −3, 765 ± 121 mmHg/s, n = 6). The time constant of left ventricular relaxation increased significantly in hearts overexpressing PL (33.4 ± 3.2 ms, n = 8) compared with uninfected hearts (18.5 ± 1.0 ms, n = 6) or hearts infected with Ad.βgal (20.8 ± 2.1 ms, n = 6). These differences in ventricular function were maintained 7 days after infection. These studies open the prospect of using somatic gene transfer to modulate overall cardiac function in vivo for either experimental or therapeutic applications.