We hypothesized that differences among individuals in reflux-induced oxidant production by esophageal squamous epithelial cells might contribute to the development of Barrett's esophagus. We studied the effects of acid and bile acids on the production of reactive oxygen species (ROS) in esophageal squamous cell lines derived from GERD patients with (NES-B3T) and without (NES-G2T) Barrett's esophagus, and in a Barrett's epithelial cell line (BAR-T). Cells were incubated with an ROS-sensitive probe and exposed to acidic media, neutral bile acid media, or acidic bile acid media. ROS were quantified in the presence and absence of DPI (an NADPH oxidase inhibitor), L-NMMA (an NO synthase inhibitor), and rotenone (a mitochondrial electron transport chain inhibitor). Acidic bile acid media induced ROS production in both squamous cell lines; however, only DPI blocked ROS production by NES-B3T cells, whereas both DPI and L-NMMA blocked ROS production by NES-G2T cells. In BAR-T cells, acidic media and acidic bile acid media induced the production of ROS; L-NMMA prevented ROS production after exposure to acidic media, whereas ROS production induced by acidic bile acid media was blocked by DPI. These studies demonstrate that there are differences between esophageal squamous cells and Barrett's epithelial cells, and between esophageal squamous cells from GERD patients with and without Barrett's esophagus in the mechanisms of oxidant production induced by exposure to acid and bile acids.