Background: Early diagnosis of esophageal cancer limited to the mucosa allows local endoscopic treatment and thereby improves prognosis. Optical coherence tomography images of normal human esophageal tissue obtained with 2 systems with light sources that provide different wavelengths (800 nm and 1275 nm) were compared with histology to determine which wavelength is best suited for detailed optical coherence tomography imaging of the esophageal wall, and to precisely localize the muscularis mucosae. Methods: Within 1 hour of surgical resection, an esophageal specimen was cleaned of excess blood with saline solution and soaked in formalin for a minimum of 48 hours. After optical coherence tomography imaging, the specimen was prepared for routine histologic assessment. To precisely localize the different layers of the esophageal wall on an optical coherence tomography image, well-defined structures within the esophageal wall were sought. Results: The 1275 nm system with 12 mm resolution was superior in terms of imaging depth. As compared with histology, the 4 μm resolution of the 800 nm system made fine detail more visible. With minimal experience, the muscularis mucosae could be recognized with either system as a hyporeflective layer with a diameter of around 180 μm. Conclusions: Based on appearance and location of morphologic landmarks, layers of normal esophageal wall, specifically, the location and extent of the muscularis mucosae, could be recognized by using both the 800 nm and 1275 nm optical coherence tomography system. Although different conditions may be operative in vivo, the present ex vivo study further verifies by precise interpretation that optical coherence tomography provides precise images of the esophageal wall. (Gastrointest Endosc 2002;56:852-7.)