Because mucosal HCl traverses the paracellular pathway before significant damage develops within esophageal epithelium, the effects of mucosal and serosal acidification were compared on Ussing-chambered rabbit esophageal epithelia. Notably serosal, but not mucosal, acidification was associated with cell necrosis, and the latter was accompanied by abolition of potential difference and short-circuit current. The reason for this difference was explored by exposing tissues serosally to ouabain, chloride-free solution, 4-acetamido-4'-isothiocyanatostilbene-2-2'-disulfonic acid (SITS), or amiloride. The results show that serosal acidification, but not ouabainization, is associated with cell necrosis and that cell necrosis induced by serosal acidification can be blocked by SITS and chloride-free solution, but not by amiloride. These findings are compatible with the hypothesis that serosal acidification is more damaging than mucosal acidification because of the greater rate with which hydrogen ions can traverse the basolateral membrane; also, the route for more rapid entry appears to involve a SITS-sensitive, chloride-dependent mechanism (e.g., Cl/HCO3 antiport).