Expression and function of cardiac ion channels exhibit postnatal developmental changes, which, however, has not yet been proven in ventricular myocytes isolated using similar techniques. In this study, ventricular myocytes were enzymatically dissociated from mouse heart at different postnatal ages (including postnatal day 0) by similar techniques using Langendorff perfusion. Whole-cell patch-clamp experiments were performed to record action potentials, I (K1), I (Kr), I (Kur), I (ss), and I (Ca,L), in ventricular myocytes freshly isolated from postnatal days 0, 7, and 14 and adult mice. Viable ventricular myocytes of day-0 mouse heart exhibited spindle-shaped appearance having cell length of approximately 50 μm, which gradually developed to a rod-shaped one having clear cross striation with cell length of approximately 120 μm (adult). The action potential duration markedly shortened, while the resting membrane potential depolarized to a small but significant extent during postnatal development. I (K1) density was maximal in postnatal day-0 ventricular myocytes and gradually decreased during development, which was accompanied by postnatal depolarization of resting membrane potential. However, I (K1) density was markedly decreased by approximately 80% in postnatal day-0 ventricular myocytes, when isolated by the chunk method. Quantitative real-time polymerase chain reaction (PCR) and western blot analyses demonstrated higher Kir2.3 expression but lower expression levels of Kir2.1 and Kir2.2 in day-0 mouse ventricles, compared with those of day-14 and adult mouse ventricles. Whereas I (Kr) exhibited marked decrease during postnatal development, I (Kur), I (ss), and I (Ca,L) exhibited postnatal developmental increase. The present cell isolation method using the Langendorff perfusion thus found that, in mouse ventricles, I (K1) exhibited postnatal developmental decrease, associated with depolarization of resting potential.