Polyethylenimine (PEI) and other polycations are good vehicles for transferring genes into the cells. In earlier reports, poly-L-lysine and protamine have been shown to improve gene delivery with cationic liposomes. In this study, PEI, combined with different cationic liposomes, was studied to determine the optimal conditions for gene delivery. The reporter genes, luciferase and green fluorescent protein, were used to transfect human HeLa, HepG2 and hepatoma 2.2.15 cells with various combinations of PEIs (0.8 and 25 kDa), poly-L-lysine (15-30 kDa), protamine and cationic liposomes. The highest expression level was achieved by using the combination of PEI 25 kDa (0.65 microg/microg of DNA, nitrogen-to-DNA phosphate (N/P) ratio=4.5) with 10 nmol of DOTAP-cholesterol (DOTAP-Chol, 1:1 w/w). This DNA complex formulation dramatically increased the luciferase expression 10- to 100-fold, which was much higher than those of other polycations alone, cationic liposomes alone or the combination. In addition, PEI/DOTAP-Chol combination had little cytotoxicity than DOTAP-Chol or other cationic liposomes alone. The effect of oligonucleotide (ODN) delivery facilitated by PEI and cationic liposomes was also studied in the hepatoma cell lines. We demonstrated an antisense ODN of p53 delivered by PEI/DOTAP-Chol combination effectively inhibited the biosynthesis of p53 protein in HepG2 (68% inhibiton) and 2.2.15 cells (43% inhibition). Thus, the large PEI could synergistically increase the transfection efficiency when combined with the cationic liposomes.