OBJECTIVE The 5'-nontranslated region (NTR) of hepatitis C virus (HCV) contains important elements that control HCV translation. The aim of this study was to determine whether antisense oligonucleotides against the NTR of the HCV genome can be targeted to inhibit HCV gene expression. METHODS Antisense oligonucleotides directed against a sequence in the internal ribosomal binding site of the NTR (anti-III) and a portion of the NTR overlapping the core protein translational start site of HCV (anti-IV) were prepared. In transient transfections of a plasmid containing a luciferase gene immediately downstream from an HCV NTR insert, oligonucleotides anti-III and anti-IV in the form of asialoglycoprotein-polylysine complexes were administered to Huh7 cells, and luciferase activity generated by cytomegalovirus (CMV) HCVluc was measured. RESULTS Anti-III inhibited luciferase activity by 75% and 99% at 0.01 mumol/L and 0.1 mumol/L, respectively. Similarly, anti-IV inhibited luciferase activity 88% and 99% at 0.01 mumol/L and 0.1 mumol/L, respectively. In cell lines stably transfected with CMV HCVluc plasmid, complexed anti-III inhibited luciferase activity in Huh7 cells by 20% at 10 mumol/L and 85% at 60 mumol/L, and was competable by an excess of asialoglycoprotein. CONCLUSIONS Antisense oligonucleotides that bind to the NTR of HCV can be targeted by receptor-mediated endocytosis, and they specifically inhibit HCV-directed protein synthesis under intracellular conditions.