Lentiviral vectors have the potential to play an important role in hemophilia gene therapy. The present study used human immunodeficiency virus (HIV)-based lentiviral vectors containing an EF1 enhancer/promoter driving human factors VIII (hFVIII) or IX (hFIX) complementary DNA expression for portal vein injection into C57Bl/6 mice. Increasing doses of hFIX-expressing lentivirus resulted in a dose-dependent, sustained increase in serum hFIX levels up to approximately 50-60 ng/mL. Partial hepatectomy resulted in a 4- to 6-fold increase (P < 0.005) in serum hFIX of up to 350 ng/mL compared with the nonhepatectomized counterparts. The expression of plasma hFVIII reached 30 ng/mL (15% of normal) but was transient as the plasma levels fell concomitant with the formation of anti-hFVIII antibodies. However, hFVIII levels were persistent in immunodeficient C57Bl/6 scid mice, suggesting humoral immunity-limited gene expression in immunocompetent mice. This study demonstrates that lentiviral vectors can produce therapeutic levels of coagulation factors in vivo, which can be enhanced with hepatocellular proliferation.