A class III antiarrhythmic agent that preferentially increases the effective refractory period without altering conduction velocity holds considerable promise for the treatment of life-threatening cardiac arrhythmias dependent on a reentrant mechanism. In the present study, the cellular electrophysiologic effects of a novel class III antiarrhythmic agent, UK-66,914, were evaluated. UK-66,914 prolonged action potential duration and extended the effective refractory period in isolated canine ventricular muscle and Purkinje fibers in a concentration-dependent manner, beginning at a threshold concentration of 0.1 microM. Analogous effects were found in isolated rabbit atrium beginning at a threshold concentration of 2 microM. At concentrations of UK-66,914 up to 20 microM there was no effect on the maximum rate of phase 0 depolarization (Vmax) or the amplitude of the action potential. In guinea pig papillary muscles. UK-66,914 at concentrations from 0.1 to 20 microM increased the effective refractory period at stimulation frequencies of 1 or 5 Hz, but did not slow conduction velocity. Therefore, UK-66,914 exhibits high selectivity for a class III antiarrhythmic effect in normal tissue. To elucidate the mechanisms responsible for the increase in effective refractory period, voltage clamp procedures were used in guinea pig ventricular myocytes. UK-66,914 reduced the amplitude of outward tail currents following depolarizing clamp steps with little effect either on the background K+ current or calcium currents, indicating that UK-66,914 selectively blocked the time-dependent potassium current. In anesthetized dogs, UK-66,914 (10 micrograms/kg to 1 mg/kg i.v.) prolonged both atrial and ventricular effective refractory periods, but in contrast to the studies performed in vitro, the minimum effective doses required to increase the effective refractory period in atria and ventricle were the same. Therefore, UK-66,914 is a potent selective class III antiarrhythmic agent, which owes its electrophysiologic profile to blockade of the time-dependent potassium current.