Background: Specialized columnar epithelium in Barrett's esophagus resembles gastric intestinal metaplasia, which selectively stains with methylene blue. Methods: We prospectively evaluated the safety, accuracy, reproducibility, cost, and diagnostic yield of methylene blue–directed biopsy in detecting specialized columnar epithelium and dysplasia in Barrett's esophagus. We performed upper endoscopy with methylene blue–directed biopsy and obtained 236 large cup biopsy specimens (145 stained, 91 unstained) from 14 patients with Barrett's esophagus of any length (Group 1) and 12 control patients. Biopsy specimens were independently examined by two pathologists unaware of the endoscopic results. Results: Methylene blue stained specialized columnar epithelium in 18 of the 26 patients, including those with intramucosal carcinoma (1), high-grade dysplasia (1), and indefinite/low-grade dysplasia (6). Methylene blue staining pattern, which was focal in 72% and diffuse in 28% of patients, was reproduced in 8 patients who had repeat staining within 4 weeks. The overall accuracy of methylene blue staining for detecting specialized columnar epithelium was 95%. The diagnostic yield of methylene blue staining for specialized columnar epithelium in “control” patients was 42%. The risk for dysplasia in stained biopsy specimens was greater than in unstained ones (odds ratio 17.7, p = .0004). Conclusions: Methylene blue mucosal staining is a safe, inexpensive, reproducible, and highly accurate method of diagnosing specialized columnar epithelium in Barrett's esophagus. (Gastrointest Endosc 1996;44:1-7.)