Background: Phospholipid species modulate cholesterol-holding capacity and, therefore, regulate bile metastability. Methods: In this study, we investigated the effect of bile salt hydrophobicity on the distribution of phospholipids among lipid particles in supersaturated model bile solutions (total lipid concentration, 9 g/dL; taurocholate/phospholipid ratio 3.0, cholesterol saturation index 1.3), by using gel permeation chromatography. Results: With an increase of bile salt hydrophobicity in the elution buffer, the uptake of cholesterol and phospholipids into bile salt micelles was increased, associated with an increased cholesterol/phospholipid molar ratio of the vesicles. In contrast, there was an inverse correlation between the hydrophobicity of the phospholipid species in the vesicles and that of bile salts in the elution buffer, suggesting that hydrophobic bile salts induced preferential uptake of hydrophobic phospholipids into bile salt micelles, while less hydrophobic phospholipids, with a relatively low cholesterol-holding capacity, remained in the vesicles. Conclusions: These data indicate that bile salt hydrophobicity regulates vesicular cholesterol metastability by modulating the hydrophobicity of phospholipids in vesicles, as well as the lipid distribution among various biliary lipid particles. [ABSTRACT FROM AUTHOR]