The purpose of the study was to prepare the pegylated liposome carrying plasmid DNA with optimal encapsulation efficiency. Plasmid DNA (pCEP4 clone 790, 10.6 kb) was entrapped in the pegylated liposome composed of neutral lipid, POPC (l-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine), cationic lipid, DDAB (dimethyl dioctadecyl ammonium bromide) and anionic lipids, DSPE-PEG 2000 (distearoyl phosphatidyl ethanolamine polyethylene glycol 2000) and DSPE-PEG 2000-maleimide by freezing/thawing method. Free plasmid DNA was separated from the encapsulated one by Sepharose CL-4B column chromatography. The DNA amount encapsulated into the pegylated liposome was increased as cationic lipid concentration, initial amount of plasmid DNA and total lipid amount were increased.