Cells that sustain double-strand breaks (DSB) can develop genomic instability, which contributes to carcinogenesis, and agents that cause DSBs are considered potential carcinogens. We looked for evidence of acid-induced DNA damage, including DSBs, in benign Barrett's epithelial (BAR-T) cell lines in vitro and in patients with Barrett's esophagus in vivo. In BAR-T cells, we also explored the mechanisms underlying acid-induced DNA damage. We exposed BAR-T cells to acid in the presence of a fluorescent probe for reactive oxygen species (ROS) and in the presence or absence of disodium 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (which prevents intracellular acidification) and N-acety-l-cysteine (a scavenger of ROS). DSBs were detected by Western blotting and immunofluorescence for histone H2AX phosphorylation and by CometAssay. During endoscopy in patients with Barrett's esophagus, we took biopsy specimens from the metaplastic mucosa before and after esophageal perfusion with 0.1 N HCl for 3 min and sought DSBs by Western blotting for histone H2AX phosphorylation. In BAR-T cells, acid exposure resulted in ROS production and caused a time-dependent increase in levels of phospho-H2AX that continued for at least 48 h. Pretreatment with disodium 4,4'-diisothiocyanatostilbene-2,2'-disulfonate or N-acety-l-cysteine prevented the acid-induced increase in phospho-H2AX levels. DSBs also were detected in biopsy specimens of Barrett's metaplasia following esophageal acid perfusion in all of 6 patients with Barrett's esophagus. Acid exposure causes DSBs in Barrett's epithelial cells through ROS produced as a consequence of intracellular acidification. These findings suggest that acid can be considered a carcinogen in Barrett's esophagus.