Crypt, but not villus, goblet cells in the ileum accelerate their secretion of mucus within 5 min following cholinergic stimulation. This study was done to determine whether the macromolecular permeability and structure of occluding junctions in the ileum are altered during accelerated secretion. Rats were injected intravenously with horseradish peroxidase followed by carbachol (250 micrograms/kg, subcutaneous) and the intestinal mucosa was fixed 3-12 min later. In control mucosa (saline-injected), peroxidase filled lateral intercellular spaces up to the occluding junctions of both crypt and villus epithelium, but did not enter occluding junctions or pass into the lumen. In 3 of 8 carbachol-stimulated rats, peroxidase was present within occluding junctions in crypt epithelium and in the crypt lumen, although all intermembrane junctional fusion sites appeared intact. Villus epithelial occluding junctions, in contrast, continued to exclude peroxidase. In freeze-fracture replicas of crypt cells prepared after carbachol stimulation, we detected no structural changes in strand networks of occluding junctions that could account for increased paracellular permeability.