T he inward rectifier current I_K1 is tightly regulated regionally within the heart, downregulated in heart failure, and genetically suppressed in Andersen syndrome. We used in vivo viral gene transfer to dissect the role of I_K1 in cardiac repolarization and maintenance of the resting membrane potential (RMP) in guinea pig ventricular myocytes. Kir2.1 overexpression boosted Ba²⁺-sensitive I_K1 by more than 100% (at –50mV), significantly shortened action potential durations (APDs), accelerated phase 3 repolarization, and hyperpolarized RMP compared with control cells (nongreen cells from the same hearts and green cells from GFP-transduced hearts). The dominant-negative Kir2.1AAA reduced I_K1 by 50–90%; those cells with less than 80% reduction of I_K1 exhibited prolonged APDs, decelerated phase 3 repolarization, and depolarization of the RMP. Further reduction of I_K1 resulted in a pacemaker phenotype, as previously described. ECGs revealed a 7.7% ± 0.9% shortening of the heart rate–corrected QT interval (QTc interval) in Kir2.1-transduced animals (n = 4) and a 16.7% ± 1.8% prolongation of the QTc interval (n = 3) in Kir2.1AAA-transduced animals 72 hours after gene delivery compared with immediate postoperative recordings. Thus, I_K1 is essential for establishing the distinctive electrical phenotype of the ventricular myocyte: rapid terminal repolarization to a stable and polarized resting potential. Additionally, the long-QT phenotype seen in Andersen syndrome is a direct consequence of dominant-negative suppression of Kir2 channel function.