The aim of our study was to develop a liposomal drug carrier system able to provide appropriate trapping efficiency of hydrophilic substance. Since majority of hydrophilic drugs are of low molecular weight, calcein was chosen as a model compound. To optimize the preparation of liposomes with regard to size and entrapment efficiency, liposomes containing calcein were prepared by different methods: conventional and modified film methods as well as proliposome method. Additionally, a dehydration-rehydration procedure was applied on the liposomes prepared by the both film methods. Ali preparations were extruded through polycarbonate membrane filters to achieve liposomes with homogenous size distribution. Regardless of the preparation method, all extruded liposomes were of mean diameter between 240 and 280 nm. However, encapsulation of calcein into liposomes differed far various preparation methods. Liposomes prepared by the modified film method could entrap more of the hydrophilic marker than those prepared by the conventional hydration method. Although the dehydration- rehydration procedure could slightly enhance the trapping of calcein into liposomes prepared by conventional film method, the extremely high encapsulation of calcein was achieved into proliposomes (> 40 %). Therefore, the proliposome method would be the right choice of preparation procedure far entrapment of hydrophilic substances.