Phospholipids have been proposed to protect the gastric mucosa by forming a proton-repellant hydrophobic layer on the gastric luminal surface, acting as a so-called gastric surfactant. The composition of this hydrophobic phospholipid layer has not previously been analysed in detail. Therefore, we measured the composition of phospholipid classes and phosphatidylcholine (PC) molecular species in gastric mucosa and mucus of rats and pigs using high resolution HPLC techniques. The predominant phospholipids of both mucosa and mucus were PC and phosphatidylethanolamine (PE). Little phosphatidylglycerol was present. The most abundant PC species of rat mucosa were PC16:0/18:1, PC16:0/18:2, PC16:0/20:4 and PC18:0/20:4. Pig mucosa also contained PC16:0/18:1, PC16:0/18:2, and PC18:0/20:4, but was poor in PC16:0/20:4. Dipalmitoyl-PC (PC16:0/16:0), the surface-active component of pulmonary surfactant, comprised only 6.42 +/- 0.33% of total PC in rat mucosa and only 5.50 +/- 1.46% of total PC in pig mucosa. Gastric mucus, isolated from both rat and pig, contained largely PC16:0/18:1 and PC16:0/18:2. The content of PC16:0/16:0 was even lower in mucus than in mucosal PC (rat 2.86 +/- 0.40%, P < 0.01; pig 1.92 +/- 0.55%, P < 0.05). We conclude that, in contrast to pulmonary surfactant, any surfactant function of the hydrophobic barrier of the stomach is unlikely to be mediated by PC16:0/16:0.