Background: Determining whether a linear catheter radio frequency (RF) ablation lesion is transmural may be difficult, especially during atrial fibrillation. We hypothesized that changes in pacing thresholds and electrogram amplitude during atrial fibrillation and sinus rhythm could be used to assess whether a radiofrequency ablation resulted in transmural necrosis. Methods: A hexapolar, linear, RF ablation catheter was positioned between the caval veins in the right atrium of seven sheep. Pacing thresholds and electrogram amplitudes during atrial fibrillation and sinus rhythm were measured before and after the application of RF energy. Sites along the linear lesion were assessed histologically. Results: The electrogram amplitude in atrial fibrillation decreased significantly more at transmural sites (unipolar recording: 33±11% transmural vs. 22±13% non-transmural, p ≤ 0.01; bipolar recording: 62±9% transmural vs. 43±15% non-transmural, p ≤ 0.01). The electrogram amplitude in sinus rhythm decreased significantly more at transmural sites (unipolar recording: 49±18% transmural vs. 15±20% non-transmural, p < 0.001; bipolar recording: 63±17% transmural vs. 42±19% non-transmural, p = 0.002). The pacing threshold increased significantly more at sites with transmural necrosis (unipolar: increased by 378±103% transmural vs. 207±93% non-transmural, p < 0.001; bipolar: 370±80% transmural vs. 259±60% non-transmural, p < 0.001). Conclusions: The amplitude of the atrial electrogram from an ablation catheter can be used to discriminate areas with transmural necrosis from those without transmural necrosis during either atrial fibrillation or sinus rhythm. Termination of atrial fibrillation may not be necessary to estimate the histologic characteristics of an ablation lesion.