The effect of sense and antisense oligodeoxynucleotides (ODNs) on hepatitis C virus (HCV) gene expression was studied to determine the role of the highly conserved 5'-untranslated region in the life cycle of the virus. It was found that antisense ODNs complementary to nucleotides (nt) 38-65, 134-175, and 312-339 in the 5' noncoding region and 341-377 in the core open reading frame efficiently blocked HCV RNA translation. Overlapping ODNs that differed by only several nucleotides showed substantially different inhibition of HCV RNA translation. Fine sequence specificity testing at nt positions 351-377 revealed that ODNs as small as a 12-mer (nt 351-363) retained a high degree (80%) of inhibitory activity compared to ODNs of longer sequences. These results suggest that there are three highly specific domains in the 5' noncoding region and a sequence immediately downstream of the HCV core initiation codon that may be critical for translation of HCV RNA. This study also provides an experimental approach for the selection of target HCV RNA sequences susceptible to antisense effects, as well as for definition of functional regions of the genome necessary for viral replication.