Cationic liposome has been studied as one of the most promising non-viral gene delivery systems. However, it has major drawbacks such as the formation of large aggregates at higher concentrations and the instability in the serum due to cationic lipid. As an alternative gene delivery system, cationic emulsion was formulated and transfection efficiency was evaluated in vitro and in vivo, in comparison with cationic liposome. Cationic emulsion was prepared with varying compositions of 3 beta [N-(N',N'-dimethylaminoethane) carbamoyl] cholesterol (DC-Chol), dioleoylphosphatidyl ethanolamine (DOPE), caster oil and Tween 80. Cationic liposome was prepared with DC-Chol and DOPE. The particle size of all the DNA/lipid complexes varied from 150 to 230 nm. The in vitro transfection efficiency of plasmid DNA was assessed by the expression of green fluorescent protein as a reporter. Of various formulations, cationic emulsion E2 (DC-Chol/DOPE/Castor Oil/Tween 80 = 0.3:0.3:0.3:0.15) and cationic liposome L3 (DC-Chol/DOPE = 0.6:0.3) showed improved transfection. DNA/E2 complexes exhibited higher transfection efficiencies (17.39+/-0.58%) in comparison with DNA/L3 complexes (11.47+/-0.59%). DNA/E2 complexes also showed a better physical stability and a stronger serum resistance than DNA/L3 complexes. Moreover, the cytotoxicity of DNA/E2 complexes was comparable to that of DNA/L3 complexes. When DNA/lipid complexes were intravenously administered, DNA/E2 complexes showed a prolonged circulation in blood and mRNA expression in various tissues compared with DNA/L3 complexes. These results suggest that cationic emulsion E2 could be a potential gene delivery system in clinical approaches because of enhanced in vivo gene transfer with low toxicity.