Most class III antiarrhythmic drugs reduce the rapidly activating component of delayed-rectifier current (IKr) without affecting the slowly activating component (IKs). Recently the novel antiarrhythmic agent azimilide (NE-10064) was reported to enhance IKs at low (nanomolar) concentrations and to block both IKr and IKs at higher (micromolar) concentrations. Further to understand the electrophysiologic effects of azimilide, we compared its effects on IKr and IKs (by using whole cell clamp techniques) and action potentials (microelectrode and perforated-patch techniques) on canine ventricular myocytes. A lower azimilide concentration (50 nM) did not enhance IKs. In contrast, a therapeutic azimilide concentration (2 microM) was equieffective in reducing IKr (300-ms isochrones) and IKs (3-s isochrones) by approximately 40% during depolarizing test pulses, as well as reducing IKr (38% decrease) and IKs (33% decrease) tail currents on repolarization. Block of IKs was independent of voltage at positive test potentials. In action-potential studies, 50 nM azimilide had no effect on the action-potential duration (APD), whereas 2 microM azimilide delayed repolarization and caused reverse rate-dependent effects on the APD. Whereas the extent of APD prolongation by azimilide was not correlated with the drug-free APD, azimilide preferentially exaggerated the APD-rate relationship of myocytes displaying the steepest APD-rate relationship under drug-free conditions. In conclusion, therapeutic concentrations of azimilide that cause comparable reduction of canine ventricular IKr and IKs exert reverse rate-dependent effects, which are dependent on the steepness of the APD-rate relationship.