The nature of the protective action of exogenous surface-active phospholipid on gastric mucosa was studied in isolated Necturus antral mucosa by measuring intracellular pH and intraepithelial potentials and resistances with a microelectrode technique. Exposure of the antral mucosa to luminal pH 2 acidified intracellular pH in surface epithelial cells by 0.6-0.3 pH units. A 20-minute pretreatment with exogenous (pulmonary) surfactanlike phospholipid completely abolished this effect. Obviously, phospholipid protected the mucosa against intracellular acidosis by decreasing the apical cell membrane conductance to H+ (and other ions), because it increased apical cell membrane resistance by +108% and total transcellular resistance by +86% but had no significant effects on paracellular or total transepithelial resistances. In mucosas exposed to three barrier-breaking agents, 10 mmol/L acetylsalicylic acid, 20% (vol/vol) ethanol, and 10 mmol/L taurocholate, at acid luminal perfusate (pH 2.0-2.5), a profound intracellular acidification of 0.9-1.3 pH units/15 min occurred. Pretreatment of the tissue with phospholipid significantly opposed intracellular acidification, but the modulatory influences on the changes in intraepithelial potentials or resistances were less conspicuous and mostly insignificant.