Cyclooxygenase-2 (COX-2) has been linked to neoplastic progression in Barrett's esophagus. Acid exposure has been shown both to activate the mitogen-activated protein kinase (MAPK) pathways and to increase COX-2 protein expression in Barrett's metaplasia, but it is not known if these effects are interrelated. We hypothesized that acid-induced activation of the MAPK pathways mediates an increase in COX-2 expression in Barrett's esophagus, and we tested this hypothesis in a Barrett's-associated adenocarcinoma cell line (SEG-1). We exposed SEG-1 cells to acidic or neutral media in the presence and absence of two MAPK inhibitors: U0126 (an ERK inhibitor) or SB203580 (a p38 inhibitor). We quantitated COX-2 protein levels using an EIA assay, and COX-2 mRNA levels using real-time PCR. We also determined how acid affects the activity of the COX-2 promoter and mRNA stability. Compared to SEG-1 cells exposed to neutral media, acid-exposed cells exhibited a 2.8-fold increase in COX-2 mRNA levels within 30 minutes. Both U0126 and SB203580 attenuated the acid-induced increase in COX-2 mRNA. Acid significantly increased COX-2 protein expression and promoter activity, and both of these effects were abolished by treatment with U0126 and SB203580. Acid exposure also stabilized COX-2 mRNA levels, an effect that was abolished by U0126 but not by SB203580. We conclude that acid increases COX-2 expression through activation of the MAPK pathways. Acid-induced activation of both ERK and p38 causes a significant increase in COX-2 promoter activity, and acid-activated ERK stabilizes COX-2 mRNA. These findings suggest potential mechanisms whereby acid reflux might promote carcinogenesis in Barrett's esophagus.