The response of the atrioventricular (AV) junction to brief intense adrenergic stimulation applied during episodes of second degree heart block achieved by acetylcholinesterase paralysis in the AV junction was examined in six dogs. Despite profound depression of AV conduction due to enhanced cholinergic activity, strong local adrenergic stimulation still readily elicited AV junctional tachycardia. Increase in cholinomimetic influences in the AV junction did not prolong transatrial or His bundle-ventricular conduction times. During AV junctional rhythm and retrograde atrial capture (n = 4), neither the sequence of retrograde atrial activation nor the atrial electrogram configurations were altered. In the two remaining dogs the AV junctional tachycardia was associated with AV dissociation. These findings suggest that the acetylcholine-induced depression of AV conduction is located in the AV node region exclusively. More important, however, is the demonstration that retrograde atrial activation originating from a pacemaker located in the AV node or immediate vicinity could actually precede the inscription of the H spike by a considerable amount of time, further suggesting that anterograde conduction from the pacemaker site to the bundle of His is far more depressed by acetylcholine than is the concomitant retrograde conduction from the pacemaker site to the atrium. Thus, inference of the origin of a subsidiary pacemaker from the P wave configuration or the relation of the A wave to the His bundle electrogram, or both, may lead to erroneous conclusions. © 1986, American College of Cardiology Foundation. All rights reserved.