Adenosine triphosphate (ATP) has potent negative dromotropic effects on the atrioventricular (AV) node, but variable effects on accessory pathway conduction have been described. The effects of an intravenous bolus injection of 8 mg ATP on accessory pathway and AV nodal conduction were determined during electrophysiologic testing with controlled atrial and ventricular rates. AV conduction was monitored during atrial or ventricular pacing at a constant cycle length, 30 msec longer than the cycle length at which block occurred. During atrial pacing antegrade block after administration of ATP occurred in 1 of 30 (3.2%) patients with accessory pathway conduction and 12 of 13 (92%) patients with AV nodal conduction (p less than 0.001). During ventricular pacing only 5 of 26 (16%) patients had accessory pathways blocked, whereas 25 of 35 (71%) patients with AV nodal conduction had block (p less than 0.001). Thus, failure of ATP to produce ventriculoatrial block identified the presence of an accessory pathway with a sensitivity of 84%, specificity of 71%, and predictive value of 72%. There was no correlation between accessory pathway properties and the effects of ATP. The effects of ATP on the AV node were concordant with the effects of a combination of verapamil and propranolol in 21 of 23 patients, suggesting that this dose ATP is an equipotent AV nodal blocker with a short duration of action. Thus, although the effects of ATP on accessory pathways and the AV node differ, block in ventriculoatrial conduction after administration of ATP cannot be used as the sole criterion to distinguish the mechanism of conduction.