We have previously demonstrated that sensitivity to interferon is different among hepatitis C virus (HCV) quasispecies simultaneously detected in same individuals and that interferon-resistant HCV quasispecies are selected during the treatment. To determine the genetic basis of their resistance to interferon, HCV genotype-1b was obtained from serum of three patients before and during interferon therapy, and their full-length nucleotide and deduced amino acid sequences were determined. Comparison of the pairs of interferon-resistant and interferon-sensitive HCV isolates in respective individuals demonstrated clusters of amino acid differences in the COOH-terminal half of the NS5A region (codon 2154-2383), which contained a common unique amino acid difference at codon 2218. Additional sequence data of the COOH-terminal half of the NS5A region obtained from six interferon-resistant and nine interferon-sensitive HCV confirmed the exclusive existence of missense mutations in a 40 amino acid stretch of the NS5A region around codon 2218 (from codon 2209 to 2248) in interferon-sensitive HCV. On the other hand, this region of interferon-resistant HCV was identical to that of prototype HCV genotype-1b (HCV-J, HCV-JTa, or HC-J4). We designated this region as the interferon sensitivity determining region. Thus, HCV genotype-1b with the prototype interferon sensitivity determining region appears to be interferon-resistant strains. The specific nature of these mutations might make it possible to predict prognostic effects of interferon treatment.