The objective of this study was to construct the pegylated liposomes containing plasmid DNA with optimal encapsulation efficiency. Plasmid DNA $(pGL2;clone;753,;{sim}6;kb)$ was encapsulated by the freeze/thawing method into liposomes composed of 1-palmitoyl-2-oleyl-sn-glycerol-3-phosphocholine (POPC), didodecyl dimethyl ammonium bromide (DDAB), distearoylphosphatidyl ethanolamine polyethylene glycol 2000 (DSPE-PEG 2000) and DSPE-PEG 2000-male-imide. The liposomes containing plasmid DNA were then extruded through two stacked polycarbonate filters with series of different pore sizes to control the liposome size. The plasmid DNA entrapped in the liposomes was separated from free plasmid DNA by Sephadex CL-4B column chromatography. The decreased pore size of polycarbonate filters resulted in the decreased size of liposomes. The encapsulation efficiency was markedly affected by the cationic lipid (DDAB) concentration, but to a low degree by the size of liposomes and by the amount of plasmid DNA.