Abundant epidemiological evidence links acid reflux to adenocarcinoma in Barrett's esophagus, but few studies have examined the cellular mechanisms by which acid promotes this neoplastic progression. We hypothesized that extracellular acid exposure causes intracellular acidification that triggers MAPK signaling and proliferation in Barrett's epithelial cells. We tested that hypothesis in a Barrett's-derived esophageal adenocarcinoma cell line (SEG-1). Methods: SEG-1 cells were exposed to varying concentrations of acid and intracellular pH (pHi) was measured by BCECF microfluorimetry. After acid exposure, Erk and p38 MAPK activation were measured by western blotting and immune complex kinase assay. Proliferation was measured by Coulter counter cell counts and an MTT incorporation assay. Results: Exposure of SEG-1 cells to solutions with a pH between 3 and 6.5 caused a rapid, reversible decrease in pHi to a level approximately equal to extracellular pH. Acid exposure caused rapid activation of both the Erk and p38 MAPKs, and also resulted in pH-dependent increases in cell number, with a maximum increase of 41% observed at pH 6.0. The MAPK activation and proliferation in SEG-1 cells induced by acid exposure could be blocked by pretreatment with DIDS, which prevents intracellular acidification by inhibiting the chloride/bicarbonate (Cl-/HCO3-) exchanger. Conclusions: In SEG-1 cells, extracellular acid exposure causes intracellular acidification which activates MAPK and causes proliferation. The magnitude of these effects is pH-dependent, and the effects can be inhibited by preventing intracellular acidification with DIDS.