This study investigated the feasibility of using liposomes to increase the hepatic delivery and antiviral efficacy of phosphorothioate antisense oligodeoxynucleotides (PS-ODN) for the in vivo treatment of hepatitis B virus (HBV) infection. Ducks infected with duck hepatitis B virus (DHBV) were used as the model. We studied the stability of an antisense PS-ODN in duck plasma, its integrity during the process of liposome entrapment, its in vivo biodistribution, plasma clearance, and excretion. In addition, the intrahepatic distribution of a labeled free and liposome-entrapped ODN was also investigated. The results of our studies show that: 1) phosphorothioate ODN remain stable during the process of liposome entrapment; 2) are stable in duck plasma for many hours; 3) are rapidly cleared from the plasma when injected intravenously; 4) intravenous injection of antisense ODNs entrapped within liposomes enhances delivery of the ODN to the liver; and 5) inhibit DHBV replication. Serum DHBV DNA levels fell rapidly, with a corresponding decrease in intrahepatic viral replicative intermediates at the end of the 5-day study period. Although inhibition of viral replication and a fall in the target protein was observed, a marked inhibition of viral replication was also observed with high doses of a random-sequence ODN. Thus, it is not certain that inhibition of viral replication was entirely through an antisense mechanism. Therefore, liposomes may be effective vehicles to improve the delivery of antisense oligonucleotides to the liver for the therapy of hepatotropic viruses.