Objective: NS5806 increases transient outward potassium current (Ito) in canine ventricular cells and augments phase 1 repolarization. Here we compare the effect of NS5806 on canine ventricular Ito to that on atrial Ito as well as on heterologously expressed putative Ito channel subunits. Methods: NS5806 (10mM) was evaluated in canine right atrial and right ventricular wedges as well as in isolated cardiomyocytes. Cloned Kv4 channels were co-expressed with putative Ito subunits in Xenopus laevis oocytes or CHO-K1 cells. Current and voltage-clamp recordings were made in the absence and presence of NS5806. Results: In ventricular wedges NS5806 increased phase 1 repolarization in epi- and midmyocardium and had minor effect on endocardial currents. In atrial preparations, the effect on phase 1 repolarization was small. To further characterize the different effects of NS5806 in different cardiac regions, Ito was measured in isolated myocytes. NS5806 increased the magnitude of Ito by 80% and 16% in ventricular epi- and endocardiomyocytes, respectively whereas in atrial myocytes, NS5806 increased peak Ito by 25% (at +40 mV) and had no effect on the sustained pedestal current, IKur. For heterologously expressed channels, NS5806 increased Kv4.3/KChIP2 peak current amplitude and slowed current decay. KCNE2, KCNE3, DPP6 and DPP10 modulated Kv4.3 currents as well as the response to NS5806, but current decay was exclusively slowed in complexes containing KChIP2. Kv1.4 was strongly inhibited by 10mM NS5806. The effects of NS5806 on currents generated by KV4.3/KChIP2/DPP6 with Kv1.4 in oocytes could reproduce those on cardiac Ito in canine ventricular myocytes Conclusions: NS5806 had differential effects on Kv4.3 channels in the presence and absence of KChIP2. Comparing the effect of NS5806 on heterologously expressed Kv4 channels and putative subunits suggests that Kv4.3/KChIP2/DPP6 as well as Kv1.4 channels contribute to canine atrial and ventricular Ito.