An iodoplatinate technique that selectively stains phospholipid was used to evaluate morphologically the effects of aspirin and 16,16-dimethyl prostaglandin E2 on canine gastric mucosa in an in vitro Ussing chamber system. The association between these morphological alterations and the changes in mucosal surface hydrophobicity as determined by contact angle analysis was also investigated. Prostaglandin (1 microgram/ml) did not alter aspirin-induced cell injury, although it reduced (45%) the detachment of damaged cells from the lamina propria. Mucus release appeared to be stimulated independently both by aspirin and prostaglandin treatment and by the blotting and drying procedure routinely performed before contact angle analysis. When an extracellular mucus gel coat was present, it contained numerous iodoplatinate-reactive vesicles and myelinated or lamellated structures. Iodoplatinate-reactive substances also appeared at the luminal surface of the mucus gel as a filamentous band. This band of reactivity was frequently seen after simultaneous treatment with prostaglandin and aspirin but only sporadically observed after aspirin treatment alone. The hydrophobic nature of the canine gastric mucosa under the above experimental conditions correlated well (r = 0.743, p less than 0.005) with the percentage of mucosal surface covered by this filamentous band, and less so with the density of iodoplatinate-reactive structures within the mucus gel. We conclude that phospholipid structures in the mucus gel may account for the hydrophobic nature of the gastric mucosal surface and the ability of damaging and "cytoprotective" agents to influence both surface wettability and barrier integrity.