Inhibition of hepatitis C virus (HCV) gene expression by antisense oligonucleotides was investigated using both a rabbit reticulocyte lysate in vitro translation assay and a transformed human hepatocyte cell expression assay. Screening of overlapping oligonucleotides complementary to the HCV 5' noncoding region and the core open reading frame (ORF) identified a region susceptible to translation inhibition between nucleotides 335 and 379. Comparison of 2'-deoxy-, 2'-O-methyl-, 2'-O-methoxyethyl-, 2'-O-propyl-, and 2'-fluoro-modified phosphodiester oligoribonucleotides demonstrated that increased translation inhibition correlated with both increased binding affinity and nuclease stability. In cell culture assays, 2'-O-methoxyethyl-modified oligonucleotides inhibited HCV core protein synthesis with comparable potency to phosphorothioate oligodeoxynucleotides. Inhibition of HCV core protein expression by 2'-modified oligonucleotides occurred by an RNase H-independent translational arrest mechanism.