The electrical properties of the transepithelial pathways of Necturaus gallbladder were studied with intracellular microelectrode techniques under control conditions and after exposure to hyperosmotic mucosal bathing media (addition of sucrose or other solutes). Doubling mucosal osmolality produces a large increase in the resistance of the intercellular shunt pathway (from 420 +/- 50 to 700 +/- 70 omega - cm2, P is less than 0.001) and a significant decrease in the resistance of the apical membrane of the cells (from 3,510 +/- 420 to 1,540 +/- 380 omega p cm2, P is less than 0.001). The basolateral membrane resistance remains unchanged. Both the apical and basolateral membranes depolarize (from 59.5 +/- 1.8 to 36.6 +/- 2.8 mV, P less than 0.0001, and from 61.8 +/- 1.7 to 32.2 +/- 3.0 mV,P is less than 0.001, respectively). The transepithelial diffusion potential resulting from NaCl concentration gradients due to the osmotic water flow does not explain the cell potential changes. Mucosal solution ion substitution experiments demonstrate increases of gk, gcl, and gna (ca. 2.5-fold, 4-fold, and 7-fold, respectively). Therefore, cell depolarization appears to be produced mainly by a reduction of apical membrane K permselectivity. The increase in the resistance of the shunt is attributable to reduction of the width of the lateral intercellular spaces, because of the osmotic serosa-to-mucosa water flow.