The absorption of phenolsulfonphthalein (phenol red) was used as a measure in vivo of intestinal permeability in anesthetized rats. A chelating agent, sodium ethylenediaminetetraacetate (NaEDTA), placed in the lumen evoked a fivefold increase in membrane permeability; at the same time the mucosal content of magnesium and calcium decreased significantly. Making either magnesium or calcium available to the luminal surface of the membrane in isotonic solution restored normal permeability and brought the cation contents above the original levels. Electron micrographs of tissues treated in vivo with NaEDTA revealed (a) rounded swellings on the microvilli in the area of the junctional complexes between adjacent epithelial cells, (b) widening of intercellular channels particularly in the region of the intermediate junctions (zonulae adhaerentes), and (c) loss of architectural detail in the region of the desmosomes (maculae adhaerentes) with separation of their dense borders. All of these alterations in fine structure could be reversed by in vivo cation replacements which reinstated normal permeability. The implications of these findings on mechanisms of fluid transport across epithelial membranes are discussed, and a working hypothesis for the role of divalent cations in membrane permeability regulation is presented.