Paroxysmal supraventricular tachycardia due to atrioventricular nodal reentry is a common arrhythmia that usually responds to medical therapy. When atrioventricular nodal reentry tachycardia is refractory to medical therapy, surgical cryoablation or endocardial catheter ablation of the His bundle has been employed to protect the ventricles from the tachycardia. However, these techniques necessitate implantation of a permanent ventricular pacemaker. The purpose of the present study was to develop a cryosurgical procedure capable of ablating the anatomic-electrophysiological substrate of atrioventricular nodal reentry by modifying, rather than ablating, atrioventricular conduction. Thirty-three adult mongrel dogs underwent either the cryosurgical procedure (n = 23) or a sham operation (n = 10). All animals were restudied immediately postoperatively (acute cryosurgery group [n = 12] and sham group [n = 10]), and 11 animals (chronic cryosurgery group) subjected to cryosurgery were studied 14 weeks postoperatively. Decremental atrial pacing and programmed premature atrial stimulation protocols were utilized to determine atrioventricular nodal conduction time, atrioventricular nodal refractory period, and the Wenckebach point before and after operation in all animals. No electrophysiological alterations were noted in the sham-operated group. In the cryosurgery groups, atrioventricular nodal conduction time, functional refractory period of the atrioventricular node, and the Wenckebach point were all significantly prolonged in the immediate postoperative period, but only atrioventricular nodal conduction time remained prolonged 14 weeks postoperatively. The potential application of the new cryosurgical procedure for the treatment of atrioventricular nodal reentry tachycardia was demonstrated in three animals that exhibited dual atrioventricular nodal conduction preoperatively but had monophasic atrioventricular conduction curves postoperatively. The results in these animals documented that the cryosurgical procedure is capable of ablating the anatomic-electrophysiological substrate necessary for atrioventricular nodal reentry tachycardia.