BACKGROUND/AIMS: Liver failure is a life threatening condition currently treated by palliative measures and, when applicable, organ transplantation. The use of a bioartificial organ capable of fulfilling the main functions of the liver would represent an attractive alternative. However, the shortage of suitable donor cells, and their limited growth ability have impeded the development of this strategy. We investigated whether lentiviral vectors allow for conditional immortalization of human hepatocytes and whether these immortalized hepatocytes could reverse lethal acute liver failure. METHODS: We exposed primary human hepatocytes to Cre-excisable lentiviral vectors coding for SV40T Antigen, telomerase, and/or Bmi-1 and tested the functionality of the resulting cell lines. Therapeutic potential of immortalized hepatocytes were tested in a murine model of acetaminophen-induced hepatic injury. RESULTS: The immortalized hepatocytes grew continuously yet were non-tumorigenic, stopped proliferating when exposed to Cre recombinase, and conserved defining properties of primary hepatocytes, including the ability to secrete liver-specific proteins and to detoxify drugs. The implantation of encapsulated immortalized human hepatocytes rescued mice from lethal doses of acetaminophen. CONCLUSIONS: Lentiviral vectors represent tools of choice for immortalization of non-dividing primary cells, and lentivirally immortalized human hepatocytes are promising reagents for cell-based therapy of acute liver failure.