A new series of gene carriers, polyethylene glycol (PEG)-grafted poly-L-lysine (PLL, mol. wt. = 25000) with three different PEG-grafted ratios (5, 10 and 25 mole%, which means 5, 10 and 25% of epsilon-amino group of PLL was modified by PEG), was synthesized. These new gene carriers, named comb-shaped PEG-g-PLL copolymer, showed a 5- to 30-fold increase in transfection efficiency compared to PLL alone on a human carcinoma cell line. It is likely that Hep G2 cells were transfected by plasmid DNA/PEG-g-PLL complexes through an endocytosis mechanism due to the fact that chloroquine increased transfection efficiency. Although Lipofectin, a cationic lipid formulation, showed slightly higher transfection efficiency than PEG-g-PLL in Hep G2 cells, our designed PEG-g-PLL demonstrated lower cytotoxicity, early gene expression and maintenance of gene expression for up to 96 h.