Phospholipid vesicles were prepared by the nonsolvent method using high-pressure CO2/water systems. The membrane properties of vesicles prepared at different pressures and temperatures were mainly characterized based on analysis of the membrane fluidity and membrane polarity, using the fluorescent probes 1,6-diphenyl-1,3,5-hexatriene and 6-dodecanoyl-N,N-dimethyl-2-naphthylamine, respectively. The CO2(liquid)/water(liquid) and the CO2(supercritical)/water(liquid) two-phase (heterogeneous) systems resulted in the formation of vesicles with high yield (ca. 85%-88%). The membrane fluidity and polarity of the vesicles were similar to those of liposomes prepared by the conventional method. It is suggested that high-pressure CO2 can be used to form an appropriate hydrophobic-hydrophilic interface where phospholipid molecules as a self-assembled membrane.