In this study, we employ antisense RNA technology to block Hepatitis B Virus (HBV) gene expression in cell culture by gene transfer as an approach to block immune recognition and pathogenic sequelae. Retroviral vectors encoding antisense and sense copies of the HBV surface antigen gene (HBsAg) were constructed, respectively. To assay the inhibition of HBV gene expression by antisense RNA, the antisense retroviral construct was co-transfected with HBV expression vector (pTHBV) in hepatoma cell line, HepG2 cells. Expression of surface antigen was assessed by a standard HBsAg assay. The results indicated that HBsAg expression was reduced (40-50%) in antisense co-transfected cells as compared to the control vector co-transfected cells. Furthermore, HepG2 was transduced with antisense retroviral vector and transfected with pTHBV. HBsAg expression was reduced 75% in the antisense retrovirus transduced HepG2 cells as compared to control vector transduced cells. The retroviral vectors developed in this study can be used to identify the target antigen of cytotoxic T lymphocytes, which contribute to the immune mediated damage in chronic HBV patients. The retroviral mediated antisense gene transfer combined with liver (or hepatocyte) transplant could also provide a molecular targeting approach for treating chronic hepatitis patients.