The resistance of the gastric mucosa to acid and peptic injury is reflected by a resistance to the back-diffusion of H+ from gastric lumen to blood1,2. The nature of this 'barrier', however, remains undefined. Using Ussing chambers, we have now studied the acid-barrier function of monolayers prepared from dispersed canine fundic chief cells3. These monolayers secrete pepsinogen in response to stimulation4. We found that, on acidification of the apical solution to pH 2, transepithelial resistance (R) increased 2.6-fold and the monolayers maintained this 1:100,000 H+ concentration gradient for more than 4 h. The addition of aspirin to the acidified apical solution caused a rapid decay in R, as did acidification of the basolateral solution to a pH < 5.5. Ouabaintreated monolayers displayed the rise in R expected with apical acidification, while potential difference (V) and short-circuit current (Isc) decreased essentially to zero, indicating impermeability to H+. However, if the integrity of the ouabaintreated monolayers was disrupted by low apical pH, H+ permeation occurred, reflected by an Isc that was dependent on the H+ gradient across monolayers. These data indicate that the apical surface of chief cells is a very tight barrier to H+ diffusion and may be an important element resisting acid-peptic injury.