Activation of muscarinic receptors in the fluid-absorptive epithelium of the Necturus gallbladder elevates cytosolic Ca2+ concentration, transiently hyperpolarizes the cell membrane voltages, and decreases the apparent fractional resistance of the apical membrane [G. A. Altenberg, M. Subramanyam, J. S. Bergmann, K. M. Johnson, and L. Reuss. Am. J. Physiol. 265 (Cell Physiol. 34): C1604-C1612, 1993]. In these studies, we show that at the peak of the hyperpolarization both apical and basolateral membrane resistances (Ra and Rb, respectively) decreased, but in 2-3 min Ra returned to control values while Rb rose to a level approximately 60% higher than control. The acetylcholine (ACh)-induced decrease in Ra is caused by activation of apical membrane maxi K+ channels secondary to elevation of cytosolic Ca2+ concentration. The increase in Rb is due to decreases in K+ and Cl- conductances. ACh had no effects on cell KCl content or water volume, although K+ conductance transiently increased. These results can be explained by the changes in basolateral membrane conductances. ACh did not alter fluid absorption. In conclusion, ACh has complex time-dependent effects on K+ and Cl- electrodiffusive permeabilities without measurable changes in cell volume or in the rate of transepithelial fluid transport.