Ambasilide (LU 47110) is a new class III antiarrhythmic drug with a unique profile of action in mammals; however, the effects on human atrial repolarization are not known. We tested the effects of ambasilide on action potentials and repolarizing potassium currents in single atrial myocytes. Ambasilide delayed all phases of repolarization in a concentration-dependent manner [i.e., 10 microM prolonged the action potential duration to 90% repolarization at 1 Hz from 217.8 +/- 34.1 to 360.6 +/- 63.0 ms (p < 0.05 vs. control)]. Action-potential prolongation was independent of the applied stimulation frequency over a range of 0.5-2 Hz; the drug therefore did not display reverse use dependence. Ambasilide produced a concentration-dependent block of the inward rectifier potassium current (IK1) and the acetylcholine-activated potassium current (IKACh) with a median effective concentration (EC50) of 6.0 and 2.3 microM, respectively. Ambasilide also led to a concentration-dependent inhibition of the transient outward current (Ito1; EC50 = 5.7 microM) and the sustained potassium outward current (ISO; EC50 = 43.6 microM). The effect of ambasilide was independent of the step voltage (in the range of +20 to +60 mV) or the applied stimulation frequency (0.5-2 Hz). Inactivation kinetics were not altered. Ambasilide is a new class III antiarrhythmic drug with a distinct profile of action. Its frequency-independent prolongation of the human atrial action potential makes this group of compounds a promising alternative to currently available class III antiarrhythmic drugs.