Acid exerts pro-proliferative effects in Barrett's-associated esophageal adenocarcinoma cells. In non-neoplastic Barrett's epithelial (BAR-T) cells, in contrast, we have shown that acid exposure has antiproliferative effects. To explore our hypothesis that the acid-induced, antiproliferative effects are mediated by alterations in the proteins that regulate the G(1)-S cell cycle checkpoint, we exposed non-neoplastic Barrett's cells to acidic media (pH 4.0) and analyzed G(1)-S checkpoint proteins' expression, phosphorylation, and activity levels by Western blot. We studied acid effects on growth (by cell counts), proliferation (by flow cytometry and bromodeoxyuridine incorporation), cell viability (by trypan blue staining), and apoptosis (by annexin V staining), and we used caffeine and small interfering RNA to assess the effects of checkpoint kinase 2 (Chk2) inhibition on G(1)-S progression. Acid exposure significantly decreased cell numbers without affecting cell viability and with only a slight increase in apoptosis. Within 2 h of acid exposure, there was a delay in progression through the G(1)-S checkpoint that was associated with increased phosphorylation of Chk2, decreased levels of Cdc25A, and decreased activity of cyclin E-cyclin-dependent kinase 2; by 4 h, a continued delay at G(1)-S was associated with increased expression of p53 and p21. Caffeine and Chk2 siRNA abolished the acid-induced G(1)-S delay at 2 but not at 4 h. We conclude that acid exposure in non-neoplastic BAR-T cells causes early antiproliferative effects that are mediated by the activation of Chk2. Thus, we have elucidated a mechanism whereby acid can exert disparate effects on proliferation in neoplastic and non-neoplastic BAR-T cells.