Skin, the biggest organ of human body, contains antigen presenting cells such as Langerhans cells (LCs) that modulate various immune responses. The skin therefore is an ideal venue to effect the transcutaneous immunization (TCI). Most current immunization procedures make use of needles and syringes for vaccine administration, which however have raised many safety concerns. To overcome the stratum corneum barrier of the skin without carrying out any skin penetration, cationic liposomes, DC-Chol/DOPE and DOTAP, were employed as vehicles for the transdermal antigen DNA delivery in this study. The optimal ratio of liposomes to DNAs for maximal transfection efficiency was determined to be 5:1 (w/w) for both formulas in BHK-21 cell transfection assays. This ratio was applied to lipoplex in tests on the dorsal skin of hair-removed mice. Reporter genes were found expressed in epidermis and spleen over 3 days. C3H/HeN mice transcutaneously immunized with the skin patch containing liposome-pCJ-3/ME (lipoplex-patch; pCJ-3/ME expressing the whole membrane and envelope protein genes of Japanese encephalitis virus (JEV)) can induce effective and protective antibodies against the infection with 50 times the 50% lethal dose (LD(50)) of JEV. The developed lipoplex-patch DNA vaccines have proven to be simple and noninvasive, by which the antibodies incurred provide marked therapeutic effects in test animals.