The interaction between natural beta-cyclodextrin and bile salts common in rat, dog and man, taurocholate, tauro-beta-muricholate, taurodeoxycholate, taurochenodeoxycholate, glycocholate, glycodeoxycholate and glycochenodeoxycholate, was studied using isothermal titration calorimetry, and the structural differences in the interaction were investigated by (1)H-ROESY NMR and molecular modeling. The beta-cyclodextrin was selected based upon its frequent use in preformulation and drug formulation as oral excipients for the solubilization of drug substances with low aqueous solubility. All the investigated bile salts possessed affinity for the cyclodextrin, though with large variations in the stability constants. The variations in the enthalpic and entropic contributions to the overall Gibbs free energy and consequently the stability constants revealed differences in the binding mode between the investigated bile salts, i.e. the bile salts with a hydroxyl group on C12 interacted differently from the bile salts without this hydroxyl group. These observations were supported by both (1)H-ROESY NMR and molecular modeling, which suggested binding on the D-ring in the steroid structure for the former and on the C-ring for the latter bile salts.