The objective of this study was to characterize immunoliposomes carrying plasmid DNA with optimal encapsulation efficiency and antibody density. Plasmid DNA was encapsulated by the freezing/thawing method into liposomes composed of POPC (1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine), DDAB (didodecyl dimethyl ammonium bromide), DSPE-PEG 2000 (distearoyl phosphatidyl ethanolamine polyethylene glycol 2000) and DSPE-PEG 2000-maleimide. The liposomes carrying plasmid DNA were extruded through two stacked polycarbonate filters, of different pore size, to control the liposome size. Then, rat IgG molecules were conjugated to the liposomes. The immunoliposomes containing plasmid DNA were separated from the free plasmid DNA and unconjugated IgG by Sepharose CL-4B column chromatography. The DNA amount encapsulated was affected by DDAB (cationic lipid) concentration, the initial amount of plasmid DNA between 10 microg and 200 microg, the total lipid amount and plasmid DNA size, but not significantly by liposome size. By varying the ratio of DSPE-PEG 2000-maleimide to IgG, the number of IgG molecules per liposome was changed significantly.