OBJECTIVE To study the specific inhibition of HBV gene expression by antisense oligonucleotide (ASON) targeted by galactosylated poly (L-lysine) (Gal-PLL). METHODS According to the results of direct sequencing of PCR amplified products, a 16 mer phosphorthioate analogue of the antisense oligonucleotide (PS-ASON) directed against the HBV U5-like region was synthesized and then linked with one liver-targeting ligand, the Gal-PLL. Using the 2.2.15 cells compared the effect of them on the expression of HBV gene. RESULTS We identified that HBV DNA in the 2.2.15 cells was from HBV with surface antigen subtype ayw2 by sequencing. The fluorescent histochemistry test indicted that Gal-PLL had a selective affinity to the rat liver tissues. A 2:1 molar ratio of the Gal-PLL to DNA optimized the complex formation. In the same experimental conditions, the inhibitory effects of HBsAg and HBeAg by PS-ASON were 70% and 58%, respectively at a concentration of 10 mumol/L, while by ligand-PS-ASON were 96% and 82%, respectively, and the amount of HBV DNA in culture supernatant and cells was depressed significantly. An unrelated sequence oligonucleotide showed no effectiveness. All the oligonucleotide had no cytotoxicity. CONCLUSIONS Antisense oligonucleotides complex with the liver-targeting ligand can be targeted to cells via asialoglycoprotein receptors resulting in specific inhibition of HBV gene expression and replication.