In order to improve the encapsulation efficiency (EN%) of a water-soluble drug in liposomes formed by the freeze-thawing (FT) method, the influence of preparation conditions was examined in detail using L-asparaginase (A-ase) as a model drug. The results were compared with those obtained by the thin film (hydration) method. It became clear that lower ionic strength in the suspension medium, gentle shaking after thawing, higher concentration of phospholipid and a relatively low concentration of A-ase were preferable to obtain higher EN% in the FT method. To confirm these results, other drugs of various molecular weights, cyanocobalamin, inulin and glucose, were encapsulated under controlled preparation conditions so as to obtain high EN%. High EN% values of from 40% to 60% (evaluated by the gel filtration method) were obtained. The influence of the phospholipid composition was also examined using phosphatidylcholine (PC), a mixture of PC and stearylamine, and a mixture of PC and dicetylphosphate. All of them gave lower EN% than yolk phospholipid (YPL). The mechanism of liposome generation is discussed on the basis of the results obtained.