Hepatitis C virus (HCV) is the major cause of non-A, non-B hepatitis worldwide. Current treatments are not curative for most infected individuals, and there is an urgent need for both novel therapeutic agents and small-animal models which can be used to evaluate candidate drugs. A small-animal model of HCV gene expression was developed with recombinant vaccinia virus vectors. VHCV-IRES (internal ribosome entry site) is a recombinant vaccinia viral vector containing the HCV 5' nontranslated region (5'-NTR) and a portion of the HCV core coding region fused to the firefly luciferase gene. Intraperitoneal injection of VHCV-IRES produced high levels of luciferase activity in the livers of BALB/c mice. Antisense oligonucleotides complementary to the HCV 5'-NTR and translation initiation codon regions were then evaluated for their effects on the expression of these target HCV sequences in BALB/c mice infected with the vaccinia virus vector. Treatment of VHCV-IRES-infected mice with 20-base phosphorothioate oligonucleotides complementary to the sequence surrounding the HCV initiation codon (nucleotides 330 to 349) specifically reduced luciferase expression in the livers in a dose-dependent manner. Inhibition of HCV reporter gene expression in this small-animal model suggests that antisense oligonucleotides may provide a novel therapy for treatment of chronic HCV infection.