In this paper the development of a new preparation method of liposomes containing a water soluble marker (fluorescein isothiocyanate-dextran (FITC-dextran) or zinc phthalocyanine tetrasulfonic acid (TSZnPc) using supercritical carbon dioxide (called "the supercritical liposome method") is described. The apparatus used consisted of two main parts: the high-pressure part, in which the lipid components 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) and cholesterol (Chol) (7:3 molar ratio) were dissolved under pressure in supercritical carbon dioxide, and a low-pressure part, in which the homogeneous supercritical solution is expanded and simultaneously mixed with the aqueous phase to yield liposomes encapsulating the water soluble marker. Addition of 7% absolute ethanol to carbon dioxide at 25 MPa and 60 degrees C and the use of a high-pressure recycling system during 30 min form the homogeneous solution with high reproducibility of both lipid components and resulted in an equal expansion profile (recovery after expansion versus time) of POPC and Chol. Incubation of the lipid components during 60 min at the above mentioned conditions generated only 3% degradation. The average size of the liposomes was about 200 nm and could not be influenced by the experimental conditions used. Optimal values for encapsulated volume (1.25 L/mol) and efficiency (20%) of the liposomes were obtained using statistical experimental design by using the water soluble marker TSZnPc and an encapsulation capillary with 5.0 cm length and 0.5 mm inner diameter. The total amount of ethanol used to obtain an encapsulation efficiency of 20% was 15-fold reduced compared to the ethanol injection method of Batzri and Korn (Biochim. Biophys. Acta 1973, 298, 1015-1019).