We report three-dimensional (3D) endoscopic microscopy findings in Barrett's esophagus, using an endoscopic optical coherence tomography (OCT) system in one patient before and in one patient after radiofrequency ablation (RFA). Findings were compared with those in a normal patient without Barrett's esophagus. In the normal patient, findings were of regular flat squamous mucosa with small subepithelial vessels and glands. In the Barrett's esophagus patient, findings were of large, densely packed glands with distortion of mucosal architecture. In the post-RFA case, findings were of a small number of isolated glands buried beneath 300-500 μm of neosquamous epithelium and lamina propria. Neosquamous epithelium is a marker of successful ablative therapy, while buried glands may have potential for dysplastic progression and are difficult to detect using conventional methods. These results indicate a potential role of 3D-OCT endoscopic microscopy for follow-up, including subsurface assessment, of ablative tratments for Barrett's esophagus.