Atrial fibrillation occurs spontaneously after bradycardia induced by acetylcholine infusion or vagal stimulation. To determine the mechanism of initiation of this tachyarrhythmia, we infused acetylcholine (5 ml, 10(-3.5) M) into Krebs-Henseleit-perfused isolated canine right atria (n = 10). Unipolar electrograms were recorded from 250 sites simultaneously during control rhythms, pacing (cycle length = 300 msec) with and without acetylcholine, and recovery of spontaneous activity. Activation sequence maps were constructed from each recording. Stable spontaneous rhythm was present in all preparations during control conditions. Activation sequence maps, recorded during continuous pacing with and without acetylcholine, demonstrated no dromotropic changes due to the acetylcholine. Focal asynchronous recovery of spontaneous activity was initiated from different sites, resulting in bigeminal or trigeminal premature depolarizations in 41 of 73 cases after infusion of acetylcholine. A reentrant tachyarrhythmia was initiated in 24 of 41 cases by the closely coupled recovery beats (A1A2 = 100 +/- 37 msec; A2A3 = 97 +/- 27 msec). The reentry was initiated by interaction of the premature impulse with regions of functional block that were a result of the cholinergically induced dispersion of refractoriness. All the tachyarrhythmias terminated spontaneously, and stable spontaneous control rhythms returned. In conclusion, the data suggest that the premature depolarizations that initiate the reentrant tachyarrhythmia are caused by the asynchronous recovery of multiple right atrial pacemakers accompanied by variable entrance block at the later depolarizing sites.