In order to obtain information for a formulation study of liposomes prepared by the freezing-thawing (FT) method, the influence of various water-soluble additives and conditions of preparation (such as storage temperature during the freezing process) on the properties of the liposomes was investigated using L-asparaginase (A-ase) as a model drug. Turbidity (A_1%), an index of the particle size, was decreased by NaCl, CaCl₂, MgCl₂ and glucose, but scarcely affected by KCl, bovine serum albumin or gelatin. The encapsulation efficiency (EN%) was decreased by all additives, but to differing extents. By optical microscopic observation, the generation of large yolk phospholipid (YPL) aggregates was found during preparation without any additive, but was not observed in the presence of NaCl. In the latter case, the mixture visibly froze at below -16°C, but both EN% and A_1% were low when it was stored at -20°C, whereas they increased remarkably when the storage temperature was lowered to below the eutectic point (-21°C) of NaCl. Both EN% and A_1% became maximum at a certain temperature and decreased at lower temperatures, and this suggests that the cooling velocity also affects them. Based on differential scanning calorimetry measurements, unfrozen water was presumed to be present around YPL particles and the amount of this water seems to affect EN% very significantly. A schematic description of the encapsulation mechanism in the FT method is proposed.