This study determined if intracolonically applied prostaglandin E₁ analogue (misoprostol) had a mucosal protective effect in rats with 4% acetic acidinduced colitis. The effects of misoprostol were compared with those of 5-aminosalicylic acid and betamethasone. A single application of 4% acetic acid induced an experimental colitis which was maximal at 2 days and showed spontaneous macroscopic and histologic healing by 12 days. Misoprostol (100 μg/kg), but not 5-aminosalicylic acid or betamethasone, administered 30 min before induction of colitis, provided macroscopic and histologic colonic mucosal protection but not protection of in vivo fluid absorption. The mucosal protective effect of misoprostol was time, dose, and diluent volume dependent. In the presence of misoprostol-induced colonic morphologic but not functional absorptive mucosal protection, in vitro unidirectional sodium and chloride flux measurements showed protection of theophylline-stimulated chloride secretion but not sodium absorption. Protection of in vivo colonic fluid absorption, in addition to morphologic protection, could be achieved when misoprostol was administered between 2 and 16 min before induction of colitis or when the highest dose (1000 μg/kg) of misoprostol was examined. We conclude that intracolonic misoprostol administration provides unique mucosal protective effects in experimental colitis.