Barrett's esophagus (BE) results from acid and bile reflux and predisposes to cancer. To further understand the mechanisms of acid- and bile-induced hyperproliferation in BE, we investigated the release of PGE₂ in response to acid or bile salt exposure. Biopsies of esophagus, BE, and duodenum were exposed to a bile salt mixture as a 1-h pulse and compared with exposure to pH 7.4 for up to 24 h, and PGE₂ release, cyclooxygenase-2 (COX-2), and protein kinase C (PKC) expression were compared. Similar experiments were also performed with acidified media (pH 3.5) alone, in the presence or absence of bisindolylmaleimide (BIM), a selective PKC inhibitor, and NS-398, a COX-2 inhibitor. One-hour pulses of bile salts or acid significantly enhanced proliferation, COX-2 expression, and PGE₂ release in BE. In contrast, the combination pulse of acid and bile salts had no such effect. Treatment with either BIM or NS-398 led to a dramatic decrease in PGE₂ release in BE explants and a suppression of proliferation. The acid- or bile salt-mediated hyperproliferation is related to PGE₂ release. Acid- and bile salt-induced induction of COX-2 and PKC may explain, at least in part, the tumor-promoting effects of acid and bile in BE.