Heart failure leads to marked suppression of the Ca²⁺-independent transient outward current (I_to1), but it is not clear whether I_to1 downregulation suffices to explain the concomitant action potential prolongation. To investigate the role of I_to1 in cardiac repolarization while circumventing culture-related action potential alterations, we injected adenovirus vectors in vivo to overexpress or to suppress I_to1 in guinea pigs and rats, respectively. Myocytes were isolated 72 hours after intramyocardial injection and stimulation of the ecdysone-inducible vectors with intraperitoneal injection of an ecdysone analog. Kv4.3-infected guinea pig myocytes exhibited robust transient outward currents. Increasing density of I_to1 progressively depressed the plateau potential in Kv4.3-infected guinea pig myocytes and abbreviated action potential duration (APD). In vivo infection with a dominant-negative Kv4.3-W362F construct suppressed peak I_to1 in rat ventriculocytes, elevated the plateau height, significantly prolonged the APD, and resulted in a prolongation by about 30% of the QT interval in surface electrocardiogram recordings. These results indicate that I_to1 plays a crucial role in setting the plateau potential and overall APD, supporting a causative role for suppression of this current in the electrophysiological alterations of heart failure. The electrocardiographic findings indicate that somatic gene transfer can be used to create gene-specific animal models of the long QT syndrome.