Ion substitution studies were carried out in the isolated perfused rat liver to define the importance of solvent drag and diffusion in bile acid-dependent bile formation. Two different methods, namely single injection (20 μmoles) and continuous infusions at 0.4, 0.8, 1.2, and 1.6 μmoles per min taurocholate (TC), were used to determine the bile acid-dependent bile flow (BADF). Both methods gave essentially the same results. Replacement of Na+ (146 mM) by 120 or 146 mM Li+ and Cl− (127 mM) by 120 mM NO3− increased BADF significantly. On the other hand, replacement of Na+ by 120 mM choline and Cl− by 120 mM isethionate decreased the BADF. The osmolarity of TC solution was not different when Na+ was replaced by 120 mM Li+ or choline and TC did not affect the osmotic activity of NaCl, LiCl, and choline-Cl differently. Thus, the observed effect of Na+ replacement on BADF is not due to any change in the osmotic activity of the secreted TC. Substitution of HCO3− by equimolar tricine also decreased BADF. Under this condition, BADF increased when NaCl was replaced by equimolar NaN03. Thus, HCO3” does not seem to be essential for TC choleresis. Since Li+ and NO3” are more permeable, and choline and isethionate are less permeable than Na+ and Cl−, respectively, these results suggest that the BADF is dependent on the permeability of the substituting cations and anions and thus support the hypothesis that solvent drag and diffusion play an important role in BADF.