Chronic psychological stress causes intestinal barrier dysfunction and impairs host defense mechanisms mediated by corticotrophin-releasing factor (CRF) and mast cells; however, the exact pathways involved are unclear. Here we investigated the effect of chronic CRF administration on colonic permeability and ion transport functions in rats and the role of mast cells in maintaining the abnormalities. CRF was delivered over 12 days via osmotic minipumps implanted subcutaneously in wild-type (+/+) and mast cell-deficient (Ws/Ws) rats. Colonic segments were excised for ex vivo functional studies in Ussing chambers [short-circuit current ( Isc), conductance ( G), and macromolecular permeability (horseradish peroxidase flux)], and analysis of morphological changes (mast cell numbers and bacterial host-interactions) was determined by light and electron microscopy. Chronic CRF treatment resulted in colonic mucosal dysfunction with increased Isc, G, and horseradish peroxidase flux in +/+ but not in Ws/Ws rats. Furthermore, CRF administration caused mast cell hyperplasia and abnormal bacterial attachment and/or penetration into the mucosa only in +/+ rats. Finally, selective CRF agonist/antagonist studies revealed that stimulation of CRF-R1 and CRF-R2 receptors induced the elevated secretory state and permeability dysfunction, respectively. Chronic CRF causes colonic barrier dysfunction in rats, which is mediated, at least in part, via mast cells. This information may be useful in designing novel treatment strategies for stress-related gastrointestinal disorders.