Thyroid hormone is known to exert important effects on cardiac repolarization, but the underlying mechanisms are poorly understood. We investigated the electrophysiological mechanisms of differences in repolarization between control guinea pigs and hypothyroid animals (thyroidectomy plus 5-propyl-2-thiouracil). Hypothyroidism significantly prolonged the rate-corrected Q-T interval in vivo and action potential duration (APD) of isolated ventricular myocytes. Whole cell voltage-clamp studies showed no change in current density or kinetics of L-type Ca²⁺ current, inward rectifier K⁺ current, or Na⁺ current in hypothyroid hearts. Dofetilide-resistant current (I_Ks) step current densities were smaller by ~65%, and tail current densities were reduced by 80% in myocytes from hypothyroid animals compared with controls. The ratio of delayed rectifier step current at +50 mV to tail current at 40 mV was significantly larger in hypothyroid cells for test pulses from 60- to 4,200-ms duration, reflecting a smaller I_Ks. Dofetilide-sensitive current (I_Kr) densities were not significantly changed. I_Ks half-activation voltage shifted to more positive voltages in hypothyroidism (29.5 ± 2.2 vs. 21.3 ± 2.7 mV in control, P < 0.01), whereas I_Kr voltage dependence was unchanged. We conclude that hypothyroidism delays repolarization in the guinea pig ventricle by decreasing I_Ks, a novel and potentially important mechanism for thyroid regulation of cardiac electrophysiology.