Somatic gene therapy of many inborn errors of metabolism may require placement of recombinant genes into liver cells in order to provide substrates and cofactors for holoenzyme activity. We report retroviral mediated transfer of recombinant genes into isolated mouse hepatocytes. Hepatocytes isolated from young mice and cultured in defined, serum-free media (SUM-3) exhibited growth (3-5 divisions) and differentiated hepatic function for 1-2 weeks in culture. Cells were infected with a recombinant retrovirus carrying the neomycin (G418) resistance gene, selected with G418 and analyzed in situ. No uninfected hepatocytes survived in 250 ug/ml G418. Infected cells expressing the recombinant neomycin resistance gene survived selection in G418, exhibited characteristic hepatocyte morphology, and continued to expresss liver specific genes alpha1-antitrypsin and phenylalanine hydroxylase. These data indicated that primary hepatocytes had been successfuly transformed with the recombinant gene and expressed the recombinant gene product. Hepatocytes containing recombinant genes can be transplanted into host animals and will continue to express differentiated function. These experiments provide a model for future gene replacement therapy in Which recombinant genes may be introduced into autologous hepatocytes and transplanted into patients in order to reconstitute a defective biological function and reverse the process of genetic disease.