Necturus gallbladder epithelial cells bathed in 10 mM HCO3/1% CO2 display sizable basolateral membrane conductances for Cl- (GClb) and K+ (GKb). Lowering the osmolality of the apical bathing solution hyperpolarized both apical and basolateral membranes and increased the K+/Cl- selectivity of the basolateral membrane. Hyperosmotic solutions had the opposite effects. Intracellular free-calcium concentration ([Ca2+]i) increased transiently during hyposmotic swelling (peak at approximately 30 s, return to baseline within approximately 90 s), but chelation of cell Ca2+ did not prevent the membrane hyperpolarization elicited by the hyposmotic solution. Cable analysis experiments showed that the electrical resistance of the basolateral membrane decreased during hyposmotic swelling and increased during hyperosmotic shrinkage, whereas the apical membrane resistance was unchanged in hyposmotic solution and decreased in hyperosmotic solution. We assessed changes in cell volume in the epithelium by measuring changes in the intracellular concentration of an impermeant cation (tetramethylammonium), and in isolated polarized cells measuring changes in intracellular calcein fluorescence, and observed that these epithelial cells do not undergo measurable volume regulation over 10-12 min after osmotic swelling. Depolarization of the basolateral membrane voltage (Vcs) produced a significant increase in the change in Vcs elicited by lowering basolateral solution [Cl-], whereas hyperpolarization of Vcs had the opposite effect. These results suggest that: (a) Hyposmotic swelling increases GKb and decreases GClb. These two effects appear to be linked, i.e., the increase in GKb produces membrane hyperpolarization, which in turn reduces GClb. (b) Hyperosmotic shrinkage has the opposite effects on GKb and GClb. (c) Cell swelling causes a transient increase in [Ca2+]i, but this response may not be necessary for the increase in GKb during cell swelling.