Intraluminally administered aspirin disrupts the gastric mucosal barrier in a pH-dependent manner and may do so by compromising the hydrophobic or nonwettable lining to the stomach. We examined the pH dependence and time-course of aspirin's effects on barrier integrity by measuring the aspirin-induced changes in surface wettability, potential difference (PD), and electrical resistance (R) of canine gastric mucosa mounted in Ussing chambers. Luminal surface hydrophobicity, as determined by contact angle (CA) measurements, and PD were reduced by acidic aspirin solutions but were not reduced by acidic solutions without aspirin (pH 2.6) or by aspirin solutions that were neutral or only mildly acidic (pH greater than 4.0). Significant reductions in CA were correlated well with the proportion of aspirin in its lipid-soluble undissociated form (r = 0.93, P less than 0.001). Acidified-aspirin solutions significantly reduced CA and PD after a 2.5-min incubation period. Significant reductions in R occurred 25 min after exposure to acidified aspirin. Morphological damage to the gastric epithelium was not apparent after a 5-min exposure to acidified aspirin but was conspicuous 30 min after aspirin treatment. Aspirin-induced reductions in CA were highly associated with decreases in mucosal PD in both the pH and time-dependent studies (r = 0.93 and r = 0.99, respectively, P less than 0.001). These data support the hypothesis that the undissociated form of aspirin compromises the protective nonwettable lining of the stomach and in doing so, disrupts the gastric mucosal barrier.