Using His bundle electrograms and the atrial (A2) and ventricular extrastimulus (V2) techniques, anterograde and retrograde refractory period studies were performed (in 9 and 12 patients, respectively) before and 10 min after intravenous infusion of phenytoin (DPH; mean plasma level, 17.3 μg/ml). DPH had no effect on the duration of the QRS complex or the H-V interval of the sinus beats; it had variable but insignificant effects on the sinus rates and the atrial, A-V nodal, and ventricular muscle refractoriness. With the use of the A2 technique, the effective refractory period (ERP) of the His-Purkinje system (HPS) could not be determined in any patient: the relative refractory period (RRP) of the HPS could be determined in 2/9 patients and shortened in both patients after DPH. With the use of the V2 technique, retrograde functional refractory period (FRP) and RRP of the HPS could be determined in all 12 patients and the retrograde ERP of the HPS in 7/12: DPH significantly shortened all these parameters (p < 0.001. < 0.001, and < 0.005, respectively). Functional refractory period of the ventriculo-atrial conduction system (VACS) could be determined in 11/12 patients during control studies (the remaining one patient had complete ventriculo-atrial block). DPH significantly shortened the FRP of the VACS in those (4) patients (Group I) in whom it was determined primarily by the HPS (p < 0.025), and had variable but insignificant effects on FRP of the VACS in the other seven patients (Group II) in whom it was determined almost exclusively by the A-V node. DPH significantly decreased the retrograde HPS conduction times of the premature impulses (V2H2 intervals) for the same coupling (V1V2) intervals (p < 0.001). It is concluded that, in the human heart, DPH exerts its most important effects on the HPS where it significantly decreases refractoriness and enhances conduction of the premature impulses. This study also demonstrates that the V2 technique is far superior to the A2, technique for evaluating the effects of drugs on refractoriness and conduction in the HPS.