Timoprazole, a substituted benzimidazole, is an antisecretory agent that inhibits gastric acid secretion by interference with (H+-K+)-ATPase. In the studies reported herein, timoprazole given orally was found to be cytoprotective for the stomach when given 30 min prior to a challenge to boiling water, ethanol, or 0.6 N HCl. Timoprazole also prevented necrosis of the mucosa and acute ulcerations induced by alcohol in the rat fundus, as evaluated by histopathology. The ED50 for cytoprotection was between 1 and 3 mg/kg of timoprazole depending on the challenge, whereas the antisecretory ED50 was approximately 12 mg/kg. Timoprazole was an active antisecretory agent when given subcutaneously (ED50 10 mg/kg), but was not cytoprotective when given by this route. Indomethacin pretreatment (5 mg/kg orally) blocked the cytoprotective activity of oral timoprazole at doses of 1 or 3 mg/kg given 30 min later. However, at higher doses of timoprazole (5 mg/kg), indomethacin did not inhibit the cytoprotective activity. The ability of high doses of timoprazole to overcome the indomethacin blocks is different than the cytoprotective activity of mild irritants, which is always blocked by indomethacin. However, when tested in vitro, timoprazole exhibited only mild inhibitory activity on both prostaglandin cyclooxygenase and 15-hydroxyl-dehydrogenase and only at high doses, suggestive of nonspecific activity.