Lipid-mediated delivery of DNA into cells holds great promise both for gene therapy and basic research applications. The primary approach to improving transfection efficiency is the design and synthesis of novel cationic lipids. Alternatively, using the synergistic effect of different cationic mixtures can provide another approach to increasing transfection efficiency. This paper describes the synergistic effect of lipids with different polarheads, central core structures and hydrophobic tails. The enhancement of cellular transfection into HEK293 cells was observed by combining two lipids having aminoglycerol and di(hydroxylethyl)amino core structures at a 1 : 1 weight ratio. Additionally, the liposome formation of these lipids with the helper lipid, 1,2-dioleoyl-propyl-3-phosphatidylethanolamine (DOPE), at the weight ratio of 1 : 1 can provide higher transfection efficiency into HEK293, MCF-7 and HeLa cells than Lipofectamine™ 2000. Our finding indicated that cationic liposomes comprised of a mixture of lipids with different polarheads, central core structures and hydrophobic tails should be very promising in liposome-mediated gene delivery in vitro and in vivo.Key words cationic liposome; synergistic effect; DNA delivery; non-viral vector.Gene therapy has gained significant attention over past two decades as an alternative method to treat genetic disorders 1,2) as well as cancers.3) One fundamental of gene therapy is the delivery system that can introduce and stabilize genetic material. Since free oligonucleotides and DNA are rapidly degraded by serum nucleases, 4) many efforts are focused on developing the carriers to protect and deliver these materials into targeted cells. Two delivery systems namely viral and non-viral vectors are currently developed to solve the problems. Viral carriers have known to be one of the most effective gene delivery methods for in vivo application. [5][6][7][8] Over one thousand gene therapy clinical trials have been completed or approved; two-thirds of which performed by viral vectors. 8)However, the limitation of viral vectors concerning toxicity, immunogenicity, scale-up procedures and their relatively small capacity for therapeutic DNA has prompted the development non-viral vectors. Delivery systems based on non-viral vectors, for example cationic lipids, dendrimers or cationic polymers, have been the focus of much recent research.9) Non-viral systems have proved to be generally less toxic or immunogenic, more easily to produce and a greater stability.Cationic liposomes are one of the most extensively studied of non-viral vectors because of their lesser immunogenic nature, ease of production and handle, and ability to deliver large pieces of DNA. Cationic liposomes, like other non-viral vectors bearing positive charge, interact with the negatively charged phosphate backbone of nucleic acids to form a compact structure and facilitate cellular uptake by endocytic routes. [10][11][12][13] Since the first application of cationic lipid in DNA delivery, 14) numerous cationic lipids...