The quaternary system of bile salt (BS)-lecithin (L)-cholesterol (Ch)-water has been mainly employed to simulate the in vitro formation of cholesterol gallstones. In this work, a quinary system of bile salt (BS)-lecithin (L)-cholesterol (Ch)-inorganic salt (Ca2+)-water was developed to study the nucleation of cholesterol crystals from a thermodynamic standpoint. The accurate concentration of each component in the freshly prepared biles and after incubation was analyzed by high performance liquid chromatography (HPLC). The micelles, vesicles and,crystals formed in the biles were characterized by quasielastic light scattering (QLS) and polarizing microscope, respectively. The results show that, under different concentrations of above five components at 37 C the model biles can finally disperse as one phase (micelles), two phases (micelles and vesicles, micelles and crystals) or three phases (micelles, vesicles and crystals). The mean diameter of BS-L micelles in the one phase region is about 5.7 nm, whereas that of Ch-L vesicles in the phase region of micelles and vesicles is about 362.4 nm. In addition, the introduction of Ca2+ will decrease the solubility of cholesterol in model biles and thus will shorten the nucleation time of cholesterol crystals, which indicates that Ca2+ plays an important role in the pathogenesis of cholesterol gallstones. (C) 2014 Elsevier B.V. All rights reserved.