Gene therapy is an attractive approach for the treatment of liver disease. We demonstrate that a so-called third-generation human immunodeficiency virus (HIV)-derived vector system can govern the efficient delivery, integration, and stable expression of a transgene into primary human hepatocytes in the complete absence of cell division. We also show that rodent hepatocytes exhibit a significant degree of resistance to HIV vector-mediated transduction, a phenotype that is particularly pronounced in murine hepatocytes and that results from a block in the immediate-early phase of infection. We finally describe a methodology, that allows very high rates of transduction through minimal in vitro manipulation, in which hepatocytes are kept in suspension and reimplanted within a few hours of harvest with a fully preserved engraftment potential. These results have immediate implications for the treatment of liver diseases by the transplantation of genetically modified hepatocytes, an approach that could be applied to a number of hereditary and acquired hepatic disorders.