High-pressure homogenization-extrusion (HPHE) is a method that can be used for downsizing large lipid vesicles with commercially available instrumentation (e.g., from Avestin Inc., Canada), which covers a full range of processing capacities from laboratory (0.5-3.5 mL) to large-scale continuous (1-1000 L/h) production. Consequently, the feasibility (at the laboratory scale) of using HPHE for producing DNA-loaded liposomes by the conventional dehydration-rehydration method was explored. HPHE-generated small unilamellar vesicles had a mean size in the range of 27-76 nm depending on the number of processing cycles and lipid (PC:DOPE:DOTAP or PC:DOPE:Ethyl-DOPC, 1:0.5:0.5, mol/mol) formulation. The size could be further regulated by the pore size (50 or 100 nm) of the extrusion membrane. Using plasmids for the V3 loop of HIV-1, and the capsid, E1 and E2 of hepatitis C, entrapment yields of 72-98.2% into dehydrated-rehydrated vesicles (DRV) were obtained over a wide range (0.309-2.5 mg) of DNA quantities. Most of the plasmid DNA was retained by liposomes even in the presence of sodium dodecyl sulfate (from 0.05% to 0.3%) and efficiently protected from nuclease-mediated degradation. Although the encapsulation process slightly decreased (in the range of 42.8-65.7%) the relative abundance of plasmid super coiled isoforms, the transfection efficiency of monkey kidney COS-7 cells with the plasmid DNA extracted from liposomes (9+/-0.4%) was similar to that of the non-treated DNA (8.7+/-0.2%), using the commercial SuperFect(R) Transfection Reagent. Also, it was found that an appreciable loss of lipid mass-either associated with the HPHE or the dehydration-rehydration steps-occurs during the liposome manufacturing process. These results at the bench scale are a useful reference for planning pilot or large-scale manufacture of DNA vaccine-containing liposomes.