We examined the mechanism by which repeated administration of indomethacin significantly delays the natural healing of experimental gastric ulcers induced in rats. Gastric ulcers were produced 5 days after injecting 20% acetic acid (0.03 ml) into the submucosal layer of the gastric wall of the antral-oxyntic border. The natural healing of the acetic acid-induced ulcers was extensively delayed by administering indomethacin (1 mg/kg) subcutaneously once daily for 2 or 4 wk. Subcutaneous administration of natural prostaglandin E2 (1 or 3 mg/kg) twice daily for 2 and 4 wk, together with indomethacin, significantly prevented the delay of ulcer healing. Prostaglandin E2 (3 mg/kg) administered twice daily for 2 wk also significantly accelerated the natural healing of the ulcers. A single administration of prostaglandin E2 (1 or 3 mg/kg) significantly reduced histamine-stimulated gastric acid secretion for 4 h in acute fistula rats with 1- or 2-wk-old ulcers, treated with or without daily indomethacin (1 mg/kg). Endogenous prostaglandin E2 levels in the gastric mucosa of normal rats were significantly reduced for at least 12 h after a single or repeated administration of indomethacin (1 mg/kg) for 2 or 4 wk. Gastric mucosal prostaglandin E2 levels in rats with ulcers (5 days after acetic acid injection) were also markedly reduced by indomethacin. This reduction significantly reverted toward control levels after administration of exogenous prostaglandin E2 (3 mg/kg). These results suggest that endogenous prostaglandin E2 plays an important role in the healing process of gastric ulcers.