Obtaining reliable information on the physical state and ultrastructure of bile is difficult because of its mixed aqueous-lipid composition and thermodynamic metastability. We have used time-lapse cryogenic transmission electron microscopy (cryo-TEM) combined with video-enhanced light microscopy (VELM) to study microstructural evolution in nucleating bile. A well- characterized model bile and gallbladder biles from cholesterol and pigment gallstone patients were studied sequentially during cholesterol nucleation and precipitation. In model bile, cholesterol crystallization was preceded by the appearance of the following distinct microstructures: spheroidal micelles (3-5 nm), discoidal membrane patches (50-150 nm) often in multiple layers (2- 10), discs (50-100 nm), and unilamellar (50-200 nm) and larger multilamellar vesicles (MLVs). The membrane patches and discs appeared to be short-lived intermediates in a micelle-to-vesicle transition. Vesicular structures formed by growth and closure of patches as well as by budding off from vesicles with fewer bilayers. MLVs became more abundant, uniform, and concentric as a function of time. In native bile, all the above microstructures, except discoidal membrane patches, were observed. However, native MLVs were more uniform and concentric from the beginning. When cholesterol crystals appeared by light microscopy, MLVs were always detected by cryo-TEM. Edges of early cholesterol crystals were lined up with micelles and MLVs in a way suggesting an active role in feeding crystal growth from these microstructures. These findings, for the first time documented by cryo-TEM in human bile, provide a microstructural framework that can serve as a basis for investigation of specific factors that influence biliary cholesterol nucleation and crystal formation.