BACKGROUND: Natural orifice transluminal endoscopic surgery (NOTES) mediastinoscopy (MED) through the esophagus has proved to be feasible in the animal model. However, injury of the adjacent pleura and pneumothorax has been reported as a frequent adverse event when using a blind access. OBJECTIVE: To assess the utility and safety of a CT-based image registration system (IRS) for navigation in the mediastinum. DESIGN: Prospective, randomized, controlled trial in 30 Yorkshire pigs. Thirty-minute MEDs were performed: 15 MEDs were performed with IRS guidance (MED-IRS), and 15 MEDs were performed with a blind access. SETTING: Animal research laboratory. INTERVENTIONS: In both groups, the mediastinum was accessed through a 10-cm submucosal tunnel in the esophageal wall. Timed exploration was performed with identification of 8 mediastinal structures. MAIN OUTCOME MEASUREMENTS: Technical feasibility, adverse events, and the number of mediastinal structures identified. RESULTS: Thirty animals weighing 31.5 ± 3.5 kg were included in this study. MED was not possible in 2 animals in the "MED with blind access" group but was possible in all MEDs performed with IRS. The mean number of identified organs was slightly higher in "with IRS-MED" (6.13 ± 1.3) than with MED with blind access (4.7 ± 2.3; P = .066). Moreover, the right atrium and vena cava were identified in more cases with IRS-MED than in MED with blind access (13 vs 3 and 15 vs 11, P = .000 and P = .03, respectively). There were 3 (23%) adverse events with IRS-MED and 4 (27%) with "MED with blind access" (P = not significant), with pneumothorax being the most frequent (2 and 3, respectively). LIMITATIONS: Nonsurvival animal study. CONCLUSIONS: This study demonstrates that the IRS system appears feasible in natural orifice transluminal endoscopic surgery MED and suggests that IRS guidance might be useful for selected procedures.