The aim of this study was to evaluate the feasibility and acute efficacy of real-time 3-dimensional transesophageal echocardiography (RT3DTEE)-guided ablation of the cavotricuspid isthmus (CVTI). The use of RT3DTEE to guide a transcatheter radiofrequency ablation procedure has never been systematically investigated. Seventy consecutive patients with CVTI-dependent atrial flutter underwent CVTI ablation. Procedural monitoring using RT3DTEE was assigned to patients who requested general anesthesia for the procedure (n = 21 [30%]). In the other 49 patients (the control group), the procedures were monitored using the standard fluoroscopic approach. Procedural time was considered as skin-to-skin electrophysiological procedure duration, not including anesthesia preparation; adequate radiofrequency ablation applications (with fixed temperature and power settings) were considered as lesions lasting ≥ 60 s. RT3DTEE allowed visualization of the CVTI and identified related structures in most patients (20 of 21); anatomic features such as long CVTI (n = 11), prominent Eustachian ridge (n = 9), prominent Eustachian valve (n = 6), septal recess (n = 8), and pectinate muscles (n = 10) were frequent. Also, RT3DTEE allowed continuous visualization of ablation catheter movement and contact. Compared with the control group, RT3DTEE was equally effective in achieving CVTI bidirectional block (100% in both groups), and no complications occurred. RT3DTEE shortened procedural time (median 73.0 min, interquartile range [IQR] 60.0 to 90.0 min, vs. median 115.0 min, IQR 85.0 to 133.0 min, p < 0.001), reduced radiation exposure (median fluoroscopy time 4.2 min, IQR 3.1 to 8.4 min, vs. median 19.3 min, IQR 12.9 to 36.4 min, p < 0.001; median fluoroscopy dose 575.4 cGy · cm(2), IQR 428.5 to 1,299.4 cGy · cm(2), vs. median 3,520.7 cGy · cm(2), IQR 1,700.0 to 6,709.0 cGy · cm(2), p < 0.001), and reduced the number of radiofrequency applications to achieve bidirectional block (median 7, IQR 6 to 10, vs. median 12, IQR 10 to 22, p = 0.007). A strong learning curve was detected by comparing procedural data between the first and last patients treated using RT3DTEE. RT3DTEE-guided ablation of CVTI was feasible, allowing real-time detailed morphological CVTI characterization as well as continuous visualization of the ablation catheter during radiofrequency ablation. This approach entailed marked reductions in procedural time, radiation exposure, and the number of radiofrequency applications.