Expression of cardiac transient outward current and inwardly rectifying K⁺ current is age dependent. However, little is known about age-related changes in cardiac delayed rectifier K⁺ current (I_K, with rapidly and slowly activating components, I_Kr and I_Ks, respectively). Accordingly, the purpose of the present study was to assess developmental changes in I_K channels in fetal, neonatal, and adult mouse ventricles. Three techniques were used: conventional microelectrode to measure the action potential, voltage clamp to record macroscopic currents of I_K, and radioligand assay to examine [³H]dofetilide binding sites. The extent of prolongation of action potential duration at 95% repolarization (APD₉₅) by a selective I_Kr blocker, dofetilide (1 μmol/L), dramatically decreased from fetal (137%±18%) to day-1 (75%±29%) and day-3 (20%±15%) neonatal mouse ventricular tissues (P<.01). Dofetilide did not prolong APD₉₅ in adult myocardium. I_Kr is the sole component of I_K in day-18 fetal mouse ventricular myocytes. However, both I_Kr and I_Ks were observed in day-1 neonatal ventricular myocytes. With further development, I_Ks became the dominant component of I_K in day-3 neonates. In adult mouse ventricular myocytes, neither I_Kr nor I_Ks was observed. Correspondingly, a high-affinity binding site for [³H]dofetilide was present in fetal mouse ventricles but was absent in adult ventricles. The complementary data from microelectrode, voltage-clamp, and [³H]dofetilide binding studies demonstrate that expression of the I_K channel is developmentally regulated in the mouse heart.