Background— In large mammals and humans, the contribution of I _Ks to ventricular repolarization is still incompletely understood. Methods and Results— In vivo and cellular electrophysiological experiments were conducted to study I _Ks in canine ventricular repolarization. In conscious dogs, administration of the selective I _Ks blocker HMR 1556 (3, 10, or 30 mg/kg PO) caused substantial dose-dependent QT prolongations with broad-based T waves. In isolated ventricular myocytes under baseline conditions, however, I _Ks block (chromanols HMR 1556 and 293B) did not significantly prolong action potential duration (APD) at fast or slow steady-state pacing rates. This was because of the limited activation of I _Ks in the voltage and time domains of the AP, although at seconds-long depolarizations, the current was substantial. Isoproterenol increased and accelerated I _Ks activation to promote APD ₉₅ shortening. This shortening was importantly reversed by HMR 1556 and 293B. Quantitatively similar effects were obtained in ventricular-tissue preparations. Finally, when cellular repolarization was impaired by I _Kr block, I _Ks block exaggerated repolarization instability with further prolongation of APD. Conclusions— Ventricular repolarization in conscious dogs is importantly dependent on I _Ks . I _Ks function becomes prominent during β-adrenergic receptor stimulation, when it promotes AP shortening by increased activation, and during I _Kr block, when it limits repolarization instability by time-dependent activation. Unstimulated I _Ks does not contribute to cellular APD at baseline. These data highlight the importance of the synergism between an intact basal I _Ks and the sympathetic nervous system in vivo.