To elucidate the critical steps during liposome preparation by detergent removal from mixed micelles, kinetics of bile salt removal were measured by monitoring radioactively labelled cholate, desoxycholate (DC) or chenodeoxycholate (CDC) in the dialysis buffer. Three different phases of bile salt interaction with lipids were found. In a first phase, a rapid removal of cholate from mixed micelles was estimated. At the end of phase I, liposome formation was completed after 4 h when using pure egg lecithin and after 3–5 h, when 30 mol% cholesterol or sphingomyelin was mixed with egg lecithin. Phases II and III showed a pronounced lower bile salt release from liposomes and were correlated with desorption from outer liposome surface and flip–flop from the inner monolayer to the outside. DC and CDC are only slowly removable from mixed micelles. Liposome formation is therefore complete only after ≈20 h. When using cholate and high lipid concentrations, formation of oligolamellar structures is due to fusion of liposomes containing a high amount of bile salt. Therefore, detergent removal has to be very fast to result in exclusively unilamellar vesicles.