Trefoil factors are small peptides found in several mammalian tissues including gut, respiratory tract and brain. Their physiological function is not well understood. Among them, trefoil factor 3 (intestinal trefoil factor) is known to be cytoprotective in the gut. However, the molecular mechanism and secondary mediators of trefoil factor 3 action are not known. In the present study, we examined whether the cyclooxygenase pathway is involved in trefoil factor 3 action. We showed that trefoil factor 3 significantly induces the production of prostaglandin E(2) and prostaglandin I(2) in IEC-18 cells (an intestinal epithelial cell line) in a dose dependent manner. Western blot and immunohistochemistry revealed that trefoil factor 3 (2.5 microM) up-regulates the expression of cyclooxygenase-2 but not cyclooxygenase-1 in IEC-18 cells. Treating cells with trefoil factor 3 (10 microM) significantly attenuated reactive oxygen species-induced IEC-18 cell injury. This effect is blocked by NS-398 (10 microM), a selective cyclooxygenase-2 inhibitor. Moreover, we demonstrated that exogenously administered carbacyclin (1 microM, a stable analogue of prostaglandin I(2)) and/or prostaglandin E(2) (1 microM) caused a significant reduction of reactive oxygen species-induced cell injury, mimicking the effect of trefoil factor 3. In summary, our results indicate that trefoil factor 3 activates cyclooxygenase-2 in intestinal epithelium to produce prostaglandin I(2) and prostaglandin E(2), which function as survival factors and mediate the cytoprotective action of trefoil factor 3 against oxidant injury.