Neonatal maternal separation (MS) predisposes adult rats to develop stress-induced mucosal barrier dysfunction/visceral hypersensitivity and rat pups to colonic epithelial dysfunction. Our aim was to examine if enhanced epithelial permeability in such pups results from abnormal regulation by enteric nerves. Pups were separated from the dam for 3h/day (days 4-20); non-separated (NS) pups served as controls. On day 20, colonic tissues were removed and mounted in Ussing chambers. Horseradish peroxidase (HRP) flux was used to measure macromolecular permeability. HRP flux was increased in MS vs NS pups. The enhanced flux was inhibited by the cholinergic muscarinic antagonist atropine and the nicotinic antagonist hexamethonium. The cholinergic component was greater in tissues from MS vs NS, suggesting that increased cholinergic activity was responsible for the MS-elevated permeability. Western blots and immunohistochemistry of colonic tissues demonstrated increased expression of choline acetyltransferase (ChAT) in MS pups, indicating greater synthesis of acetylcholine. Since a previous study indicated that corticotrophin-releasing factor (CRF) mediates barrier dysfunction in MS pups, we examined if the two pathways were linked. In MS tissues, non-selective CRF receptor antagonism inhibited the enhanced flux, and addition of atropine did not produce further inhibition. Using selective receptor antagonists, we identified that CRF receptor 2 (CRF-R2) was involved in mediating this effect. These findings suggest that CRF, via CRF-R2, acts on cholinergic nerves to induce epithelial barrier dysfunction. Our study provides evidence that MS stimulates synthesis of acetylcholine, which together with released CRF, creates a condition conducive to development of epithelial barrier defects.