Although computerized mapping has enabled the intraoperative delineation of global ventricular activation from a single complex of ventricular tachycardia (VT), beat-to-beat reproducibility of isochronic maps has not been defined. To determine the reliability of single-beat analysis, epicardial and transmural ventricular electrograms during 6 consecutive VT cycles were analyzed in 10 patients during intraoperative mapping of sustained monomorphic VT. Bipolar electrograms were recorded simultaneously using sock and needle electrodes from up to 96 epicardial and 156 transmural sites. In each patient, at each electrode site, local activation time, electrogram duration and morphology were compared over 6 consecutive beats. A total of 9,816 electrograms were analyzed. For each patient, the isochronic activation map during VT was reproducible. Mean beat-to-beat variations in local epicardial and transmural activation times were only 1.7 ± 1.7 and 2.04 ± 1.9 ms, respectively (difference not significant). Moreover, electrogram duration did not vary significantly. Mean variations in epicardial and transmural electrogram durations were 2.1 ± 1.8 and 1.4 ± 1.9 ms, respectively (difference not significant). There were only 2 instances of 2:1 conduction failure; both occurred intramurally and adjacent to a site of VT origin. Thus, transmural ventricular activation during sustained monomorphic VT is reproducible regardless of electrode site or electrogram duration. These results demonstrate that analysis of a single beat of VT is a reliable and expedient method to delineate ventricular activation during intraoperative computerized mapping for the purpose of clinical decision-making in patients with sustained monomorphic VT.