BACKGROUND AIMS: The molecular mechanism underlying epithelial metaplasia in Barrett s esophagus remains unknown. Recognizing that Hedgehog signaling is required for early esophageal development, we sought to determine if the Hedgehog pathway is reactivated in Barrett s esophagus, and if genes downstream of the pathway could promote columnar differentiation of esophageal epithelium. METHODS: Immunohistochemistry, immunofluorescence, and quantitative real-time polymerase chain reaction were used to analyze clinical specimens, human esophageal cell lines, and mouse esophagi. Human esophageal squamous epithelial (HET-1A) and adenocarcinoma (OE33) cells were subjected to acid treatment and used in transfection experiments. Swiss Webster mice were used in a surgical model of bile reflux injury. An in vivo transplant culture system was created using esophageal epithelium from Sonic hedgehog transgenic mice. RESULTS: Marked up-regulation of Hedgehog ligand expression, which can be induced by acid or bile exposure, occurs frequently in Barrett s epithelium and is associated with stromal expression of the Hedgehog target genes PTCH1 and BMP4. BMP4 signaling induces expression of SOX9, an intestinal crypt transcription factor, which is highly expressed in Barrett s epithelium. We further show that expression of Deleted in Malignant Brain Tumors 1, the human homologue of the columnar cell factor Hensin, occurs in Barrett s epithelium and is induced by SOX9. Finally, transgenic expression of Sonic hedgehog in mouse esophageal epithelium induces expression of stromal Bmp4, epithelial Sox9, and columnar cytokeratins. CONCLUSIONS: Epithelial Hedgehog ligand expression may contribute to the initiation of Barrett s esophagus through induction of stromal BMP4, which triggers reprogramming of esophageal epithelium in favor of a columnar phenotype.