The onset of recurrent or sustained atrial fibrillation (AF) is common during electrophysiological (EP) studies of accessory pathways (AP). We report our experience in patients with Wolff-Parkinson-White (WPW) syndrome in whom AF with rapid antegrade conduction over the AP occurred during an EP study and mapping and ablation were done during sustained AF, as compared to patients ablated during sinus rhythm. The study group consisted of 18 patients (group 1) with WPW syndrome who underwent catheter ablation during pre-excited AF. Two hundred and sixty-three patients, comparable for clinical characteristics, whose manifest APs were ablated under sinus rhythm formed the control group (group 2). Bipolar electrogram criteria recorded from the ablation catheter showing early ventricular activation relative to the delta wave on the surface ECG and AP potentials preceding the onset of ventricular activation were used as targets for ablation. Clinically documented atrial fibrillation was significantly more frequent and antegrade ERP of AP was significantly shorter in group 1 than in group 2 (39% vs 14%, P=0.014 and 268+/-37 vs 283+/-16, P<0.001, respectively). Procedure-related variables, acute success rates (17/18 [94%] in group 1, 251/263 [95%] in group 2; P>0.05) and late recurrence rates (0/18 [0%] in group 1 vs 5/263 [2%] in group 2; P>0.05) during a mean follow-up of 25+/-9 months (range 8-52 months) did not differ significantly. Our results show that both right- and left-sided accessory pathways can be mapped and ablated safely during pre-excited AF without delay, and that acute success and recurrence rates and long-term follow-up results are similar to those of pathways ablated during sinus rhythm.