BACKGROUND & AIMS: It is not clear why only a minority of patients with gastroesophageal reflux disease (GERD) develop Barrett's esophagus. We hypothesized that differences among individuals in molecular pathways activated when esophageal squamous epithelium is exposed to reflux underlie the development of Barrett's metaplasia. METHODS: We used esophageal squamous cell lines from patients who had GERD with Barrett's esophagus (normal esophageal squamous [NES]-B3T and NES-B10T) and without Barrett's esophagus (NES-G2T and NES-G4T) to study effects of acid and bile salts on expression of the CDX2 gene. Bay 11-705, Ad5 inhibitor kappaB(IkappaB)alpha-SR, and site-directed mutagenesis were used to explore effects of nuclear factor-kappaB (NF-kappaB) inhibition on CDX2 promoter activity; DNA binding of the NF-kappaB subunits p50 and p65 was assessed by chromatin immune-precipitation. RESULTS: Acid and bile salts increased CDX2 messenger RNA (mRNA), protein, and promoter activity in NES-B3T and NES-B10T cells, but not in NES-G2T or NES-G4T cells. Inhibition of NF-kappaB abolished the increase in CDX2 promoter activity. Increased CDX2 promoter activity was associated with nuclear translocation of p50, which bound to the promoter. We found CDX2 mRNA in 7 of 10 esophageal squamous biopsy specimens from patients with Barrett's esophagus, but in only 1 of 10 such specimens from patients who had GERD without Barrett's esophagus. CONCLUSIONS: Acid and bile salts induce CDX2 mRNA and protein expression in esophageal squamous cells from patients with Barrett's esophagus, but not from GERD patients without Barrett's esophagus. We speculate that these differences in acid- and bile salt-induced activation of molecular pathways may underlie the development of Barrett's metaplasia.