The functional importance of the "mucus-bicarbonate barrier" in protecting the gastric mucosa against injury by acid or pepsin was examined using an ex vivo gastric chamber preparation in the rat. Conditions were created such that the effectiveness of a mucus-stabilized pH gradient on the mucosal surface would be minimized. Thus solutions of hydrochloric acid of pH 1.3 or 0.8 were applied to the mucosal surface and continually stirred for 60 min. By use of an antimony pH microelectrode, these concentrations of acid were shown to dissipate the pH gradient on the mucosal surface within 5 min. The effects of addition of the mucolytic agents, pepsin or N-acetylcysteine, to both acid solutions were also assessed. Finally, the ability of the mucosa to resist injury by acid after disruption of the surface epithelium (with hypertonic saline) was examined. Exposure to the acid solutions, with or without added mucolytic agents, was without damaging effects on the mucosa, as assessed macroscopically, histologically, or by measurement of transmucosal potential difference and luminal protein concentration. Conversely, disruption of the surface epithelium rendered the mucosa significantly more susceptible to the damaging actions of acid. These results therefore demonstrate that under conditions in which a pH gradient on the mucosal surface was no longer detectable, the mucosa was resistant to injury by acid. In the undamaged rat stomach, therefore, the mucus-bicarbonate barrier may be functionally redundant.