To examine whether hepatocytes transplanted in the spleen can function as an ectopic liver, we performed hepatocyte transplantation in rats that were rendered anhepatic. Total hepatectomy was performed by using a novel single-stage technique. Following hepatectomy, Group 1 rats (n β«Ψβ¬ 16) were monitored until death to determine survival time without prior intervention. Group 2 anhepatic rats (n β«Ψβ¬ 20) were sacrificed at various times to measure blood hepatocyte growth factor (HGF) and transforming growth factor β€1 (TGF-β€1) levels. Group 3 (n β«Ψβ¬ 16) rats received intrasplenic injection of isolated hepatocytes (2.5 Ψ 10 7 cells/rat) followed by total hepatectomy after 3 days. Group 4 (n β«Ψβ¬ 12) sham-transplanted rats received intrasplenic saline infusion, and after 3 days they were rendered anhepatic. Group 2, 3, and 4 rats were maintained on daily Cyclosporine A (10 mg/kg; intramuscularly). Group 1 anhepatic rats survived for 22.4 Ψ 5.2 hours (standard deviation). The anhepatic state was associated with a progressive and statistically significant rise in blood HGF and TGF-β€1 levels. Rats that received hepatocyte transplantation before total hepatectomy had a significantly longer survival time than sham-transplanted anhepatic controls (34.1 Ψ 8.5 vs. 15.5 Ψ 4.8 hrs, P F .01). Additionally, at 12 hours post-hepatectomy, transplanted rats had significantly lower blood ammonia, prothrombin time, international normalized ratio, and TGF-β€1 levels when compared with sham-transplanted controls. In conclusion, intrasplenic transplantation of allogeneic hepatocytes prolonged survival, improved blood chemistry, and lowered blood TGF-β€1 levels in rats rendered anhepatic. (HEPATOLOGY 1998;28: 1365-1370.) Transplantation of isolated hepatocytes (HcTx) has been shown to ameliorate, at least partially, specific enzymatic defects 1-4 and provide temporary support to animals with experimentally induced liver failure. 5-10 Preliminary results Abbreviations: HcTx, hepatocyte transplantation; FHF, fulminant hepatic failure; TH, total hepatectomy; HGF, hepatocyte growth factor; TGF-β€1, transforming growth factor β€1; IVC, inferior vena cava.