Different methods and conditions for ATP incorporation into PEGylated liposomes were compared in order to obtain a preparation with a maximized ATP content. Such a preparation may find the application for the in vivo treatment of ischemic tissues suffering from an insufficient ATP supply. Several different methods of liposome preparation and purification were used and HPLC was employed to determine the concentration of ATP in the liposomes. Thin lipid film hydration produced vesicles with the lowest ATP encapsulation (ca. 5 mol%). A pH gradient method yielded liposomes with ca. 10 mol% of ATP. Reverse phase evaporation and freezing-thawing methods resulted in a maximum entrapment of ATP on the level of 36-38 mol%. The freezing-thawing method was chosen for further investigation because of its simplicity and absence of a need to use organic solvents. The separation of the non-entrapped ATP by gel-filtration, centrifugation or dialysis yielded virtually identical liposomal preparations. The incorporation of PEG (as PEG-distearoyl phosphatidylethanolamine, PEG-DSPE) into the liposomal membrane decreases the quantity of the entrapped ATP (from 38 mol% for liposomes with 0.5 mol% of PEG-DSPE to only 17 mol% for liposomes with 5 mol% of PEG-DSPE).