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Hepatocellular transplantation (HCT) could provide a therapeutic alternative to orthotopic liver transplantation (OLT) in the treatment of hepatic metabolic defects and experimental hepatic failure[1-4]. Under appropriate conditions, the engrafted liver cells can continue to express liver-specific functions for an indefinite period of time. The major limitation of many animal studies in HCT is that, since the donor hepatocytes are often indistinguishable from those of the host, it has often been difficult to demonstrate a clear correlation between engraftment and the therapeutic effect. In order to verify engraftment dependent on the therapeutic response, a recombinant retroviral vector carrying marker genes is used to label the donor hepatocytes[5,6]. The vector is capable of transducing hepatocytes, integrating gene stably into the genome and directing expression. Efficient retroviral-mediated gene transfer has introduced the possibility of targeting genetic markers to hepatic cells and somatic gene therapy for liver diseases[7-11]. Stable integration and expression of retroviral genes is dependent upon active division of the infected cell[9-13]. Although hepatocytes maintain growth potential in vivo and are capable of substantial regeneration following partial hepatectomy, their ability to grow in culture is quite limited. In the present study, we explored the optimal culture system for hepatocyte proliferation and the potential for retroviral-mediated gene transfer into primary hepatocytes. We successfully demostrated the efficient and stable transduction of primary culture of adult rat hepatocyte by replication of defective retrovirus carrying β-gal gene and NeoR gene.