Previous studies suggested that transient outward potassium current (Ito) plays an important role only during the early fast phase of repolarization, modulating the plateau phase potential, but its direct influence on the total repolarization is negligible. In this study we took advantage of the observation that at high (100 μM) concentration chromanol 293B blocks not only the slow delayed rectifier (IKs), but also largely inhibits Ito. Therefore, in the presence of full IKs block (by L-735,821) chromanol 293B induced change of action potential (AP) and of ionic currents can be attributed to its effect on Ito. Based on this assumption, the aim of the present study was to investigate the role of Ito in cardiac ventricular repolarization applying the standard microelectrode and patch-clamp techniques. Chromanol 293B (100 μM), in the presence of full IKs block (100nM L-735,821), lengthened the AP repolarization in both endocardial and epicardial dog right ventricular preparations. The next set of experiments was carried out in the presence of 100 nM dofetilide and 1 μM BayK 8644 to impair the repolarization reserve of the preparations. In these measurements chromanol 293B excessively lengthened the AP compared to that found after application of another IKs inhibitor, HMR-1556, which can be attributed to the Ito blocking effect of chromanol 293B. In single dog ventricular myocytes the inactivation kinetics of Ito current was biexponential (rapid phase: 3.89±0.13 ms, amplitude: 2222±239 pA; slow phase: 23.5±2.5 ms, amplitude: 172±33 pA; n=12, test potential: 20 mV, means±S.E.M.). The amplitude and the time constant of the slow phase of Ito imply an important role of the slowly inactivating Ito in the plateau phase of the action potential. The little overlap of the inactivation and activation curves over the potential range of 0 to –40 mV proposes that a small fraction of these ion channels may reactivate in this potential range (Ito “window current”). The Ito current during the AP plateau and repolarization phases was measured as chromanol 293B sensitive current in left ventricular myocytes isolated from dog hearts using an AP waveform for the command potential. It was found that Ito carries significant outward current in the plateau and also in the later phase of repolarization of the AP. We concluded that the transient outward potassium current may influence not only indirectly, by modulating the plateau potential, but also directly the ventricular repolarization in the heart.