The role of calcium in the biliary and intestinal milieu may be quite central, forming physiologically important complexes of bile salts. The binding of Ca2+ to bile salts in aqueous solution, in particular to glycocholate and taurocholate, has been investigated by using Ca2+-specific electrodes. The reported dissociation constants are surprising since they suggest that the sulfonate-bearing taurocholate has a greater affinity for Ca2+ than the carboxylate-bearing glycocholate. Hydroxyl involvement in Ca2+ binding to glycocholate has also been suggested (Moore, E. Hepatology 1984, 4, 228S-243S). This may bring geometrical contraints on the molecule, which may result in a thermodynamic preference for binding in the case of taurocholate vs. that in the glycocholate system. In the present study we have used a paramagnetic NMR approach in which the lanthanide ion Dy3+, an isomorphous replacement for Ca2+, causes concentration-dependent stereospecific changes in the bile salt 1H chemical shifts. The dysprosium-induced effects have been modulated by the addition of CaCl2 or NaCl. Analysis of the data by nonlinear computer programs has enabled calculation of the metal-bile salt dissociation constants for Dy3+, Ca2+, and Na+ complexes. For each metal ion the dissociation constant of the metal-bile salt complex was 2–6 times larger for taurocholate than for glycocholate. These data are consistent with the idea that the Ca2+ ion has a greater affinity for the carboxylate group than for the sulfonate group. The intrinsic shifts of 1H resonances, which are sensitive to changes in the position of the Dy3+ ion, decrease gradually for protons the further they are from the carboxylate end, indicating metal binding to the carboxylate group. These intrinsic shifts are unaltered in the presence of CaCl2 or NaCl, suggesting that Ca2+ competes for binding to the anionic group and not to any other site on the bile salt molecule. © 1986, American Chemical Society. All rights reserved.