Abstract We studied the effect of the prostaglandin synthesis inhibitor, indomethacin, on pentagastrin-associated enhancements of gastric mucosal defense mechanisms, using a microfluorometric technique in which mucus gel thickness, intracellular pH (pH i), gastric mucosal blood flow, and acid secretion were measured simultaneously in vivo. Intravenous infusion with pentagastrin (80 μg/kg/h) increased mucus gel thickness, induced a hyperemic response to luminal acid, and enhanced pH i homeostasis during acid superfusion. Indomethacin, (5 mg/kg, IP) did not alter the effects of pentagastrin on acid output, mucus gel thickness, mucosal blood flow, and pH i homeostasis. Indomethacin alone did not affect any of these measures. We conclude that the enhancement of gastric defense mechanisms due to pentagastrin is independent of prostaglandin synthesis.