Clinical studies have demonstrated that retrograde conduction of a premature beat through the His-Purkinje system can be facilitated by atrioventricular sequential pacing. Several possible mechanisms of facilitation have been proposed. No studies, however, have shown the occurrence of this phenomenon or its mechanism in isolated Purkinje fibers. The present study demonstrated that facilitation of conduction of a premature beat can indeed occur in isolated canine Purkinje fibers during sequential pacing. When a premature beat showed conduction delay during unidirectional pacing, its conduction consistently improved during sequential pacing. This improvement of conduction was related to a greater membrane recovery of a portion of the Purkinje fiber, i.e., the portion that was pre-excited by the sequential mode of stimulation. These findings suggest that an important mechanism of the facilitation of conduction observed clinically may be similar; i.e., pre-excitation and consequent earlier recovery from refractoriness of portions of the His-Purkinje system during atrioventricular sequential pacing.