Objective: NS5806 activates the transient outward potassium current (Ito) in canine ventricular cells. We compared the effects of NS5806 on canine ventricular versus atrial. Methods: NS5806 (10 M) was evaluated in arterially-perfused canine right atrial and ventricular wedges. Atrial and ventricular epi- and endocardial cells were isolated by enzymatic dissociation. Current and voltage-clamp recordings were made in the absence and presence of NS5806. Expression of ionchannel subunit mRNA in atrial and in ventricular tissue was compared by Real Time PCR. Results: In ventricular wedges NS5806 augments phase 1 repolarization in epi- and midmyocardial cells. A minor effect on conduction and upstroke velocity also was observed. In contrast, application of NS5806 to atrial preparations slowed upstroke velocity and reduced excitability, consistent with sodium channel block. In isolated ventricular myocytes, NS5806 increased the magnitude of Ito by 80% and 16% in epi and endo, respectively (at +40 mV). In atrial myocytes, NS5806 increased peak Ito by 25% and had no effect on the sustained pedestal current, IKur. INa density in atrial myocytes was nearly 100% greater than in endocardial myocytes. NS5806 caused a negative shift in the steady-state mid-inactivation potential for both cell types (73.9±0.27 to -77.3±0.21mV for endocardial and -82.6±0.12 to -85.1±0.11mV for atrial cells). The shift in the mid-inactivation potential resulted in a reduction of INa in both cell types. However, the more negative mid-inactivation potential in atrial cells suggests that atrial cells lose excitability at more depolarized voltages than endocardial cells which may explain the greater reduction of excitability in atrial vs ventricular wedges by NS5806. Conclusion: NS5806 produces a prominent augmentation of Ito with little effect on INa in the ventricles, but a potent inhibition of INa with little augmentation of Ito in atria. The different response to NS5806 could suggest different subunits contribute to Ito as well as INa channels the ventricle and atria.