The thermodynamics of molecular self-assembling of an anionic biosurfactant, sodium cholate (NaCA) and its mixtures with sodium dodecyl sulfate (SDS) in aqueous solution have been investigated by isothermal titration calorimetry (ITC), along with fluorescence and conductivity measurements. Different critical concentrations were obtained by these three techniques - critical pre-micelle concentration (cmc(pre)) and critical micelle concentration (cmc) for pure NaCA, and critical micelle concentrations (cmc(mix)) for the mixed systems with differently initial SDS concentrations. Importantly, ITC allowed us to directly measure the enthalpy changes of pre-micelle formation (DHpremic = (-0.28 +/- 0.02) kJ.mol(-1)) and of micelle formation (Delta H-mic = (-1.76 +/- 0.05) kJ.mol(-1)) for pure NaCA as well as the enthalpies for micellization for the mixed systems NaCA/SDS. The non-ideality of the mixed surfactant solution was evaluated in terms of interaction parameters and excess enthalpies that were calculated in the light of Clint's and Rubingh's models. It was found that there is an obvious synergistic effect in the NaCA/SDS mixed system. From all these results we can ascribe the strong interaction between the same charge surfactants NaCA and SDS to the structural difference in their hydrophobic moieties. In fact, the flexible alkyl chains of SDS and the non-planar hydrophobic beta-faces of NaCA tend to have a more compact packing than pure NaCA. (C) 2016 Elsevier Ltd. All rights reserved.