INTRODUCTION The influence of the autonomic nervous system on the pathogenesis of complex fractionated atrial electrograms (CFAE) during atrial fibrillation (AF) is incompletely understood. This study evaluated the impact of pharmacological autonomic blockade on CFAE characteristics. METHODS AND RESULTS Autonomic blockade was achieved with propanolol and atropine in 29 patients during AF. Three-dimensional maps of the fractionation degree were made before and after autonomic blockade using the Ensite Navx system. In 2 patients, AF terminated following autonomic blockade. In the remaining 27 patients, 20,113 electrogram samples of 5 seconds duration were collected randomly throughout the left atrium (10,054 at baseline and 10,059 after autonomic blockade). The impact of autonomic blockade on fractionation was assessed by blinded investigators and related to the type of AF and AF cycle length. Globally, CFAE as a proportion of all atrial electrogram samples were reduced after autonomic blockade: 61.6 +/- 20.3% versus 57.9 +/- 23.7%, P = 0.027. This was true/significant for paroxysmal AF (47 +/- 23% vs 40 +/- 22%, P = 0.003), but not for persistent AF (65 +/- 22% vs 62 +/- 25%, respectively, P = 0.166). Left atrial AF cycle length prolonged with autonomic blockade from 170 +/- 33 ms to 180 +/- 40 ms (P = 0.001). Fractionation decreases only in the 14 of 27 patients with a significant (>6 ms) prolongation of the AF cycle length (64 +/- 20% vs 59 +/- 24%, P = 0.027), whereas fractionation did not reduce when autonomic blockade did not affect the AF cycle length (58 +/- 21% vs 56 +/- 25%, P = 0.419). CONCLUSIONS Pharmacological autonomic blockade reduces CFAE in paroxysmal AF, but not persistent AF. This effect appears to be mediated by prolongation of the AF cycle length.