Time-sequential enzymatic determination of cholesterol (CH) crystals harvested by ultrafiltration, and concomitant polarizing light microscopy observations corroborated the striking importance of the bile salts (BS) species in determining CH crystals formation rate from supersaturated model biles incubated in vitro. The more hydrophilic tauroursodeoxycholate, taurohyocholate, glycohyocholate, taurohyodeoxycholate, glycohyodeoxycholate and glyco-3 alpha, hydroxy-6 oxo-5 beta-cholanate inhibited CH precipitation through the formation of a stabilized liquid-crystalline phase. In contrast, in all hydrophobic systems (taurine (T) and glycine (G) conjugates of cholate (C), deoxycholate (DC) and chenodeoxycholate (CDC)), CH crystals precipitated with time. When crystallized CH concentrations were plotted vs. time, the figures showed a sigmoidal pattern, consistent with the transition from metastable systems to stable equilibrium states. Over the equilibration period, the nucleation kinetics (as inferred from enzymatic measurements) and all crystallization events (as microscopically observed) were both shifted in time, depending on the BS species: they were earliest in CDC systems, then in DC systems, and finally in C systems. In the latter, the delay was clearly due to the formation of a transient labile liquid-crystalline phase. G-conjugation also induced a significant delay in CH precipitation, compared to T-conjugation. At last, maximum crystallized CH concentrations at equilibrium were in the decreasing order: C > CDC > DC and T-conjugates > G-homologues. All data are discussed in connection with BS hydrophobicities, with predictions from the phase equilibria of aqueous biliary lipid systems and with new insights into CH crystal habits.