The Freeze-thawing (FT) technique was adopted in order to improve the encapsulation efficiency of timolol maleate in liposomes. The results were compared with those obtained by the conventional hydration (HY) method. The FT liposomes showed higher encapsulation efficiency and lower permeability properties than the HY liposomes. The influence of surface charge, drug and lipid concentration, presence of sucrose and ionic strength of the medium the encapsulation efficiency of FT liposomes was also investigated. The results revealed that the formation of large liposomes by this technique which probably results from fusion of vesicles is strongly inhibited by increasing the ionic strength, by the presence of sucrose and by increasing the lipid concentration of liposomes.