The response rate to interferon alfa (IFN-α) in patients infected with hepatitis C virus (HCV) genotype 1 isolates is poor. A region associated with sensitivity to IFN has been identified in subtype HCV- 1b isolates from Japanese patients in the carboxyterminal half of the nonstructural protein NS5A (between codon 2209 and 2248). HCV-1b isolates with at least four amino acid changes in this region compared with the HCV-1b prototype sequence were sensitive, whereas isolates identical to the prototype sequence were resistant to IFN-α. Patients infected with HCV-1b isolates carrying 1 to 3 mutations in NS5A2209-2248 showed an intermediate response pattern. Because of the large geographical differences observed for HCV it is unknown whether this putative IFN-α sensitivity determining region is also predictive for European isolates. We analyzed 32 patients chronically infected with HCV-1a or HCV-1b isolates who were treated with 3 million units of recombinant IFN-α three times per week for 1 year. Before initiation, during, and after treatment serum HCV-RNA levels were assessed by a quantitative reverse-transcription polymerase chain reaction (RT-PCR) assay. The amino acid sequence of NS5A2209-2248was determined by direct sequencing of the PCR-amplified HCV genome and was compared with the reference sequence HCV-J. In patients chronically infected with subtype HCV-1a or HCV-1b the initial or sustained response to IFN-α was not related to the number of amino acid substitutions in the NS5A2209-2248 region. In addition, the number of amino acid changes in NS5A2209-2248 was not related to pretreatment HCV-RNA serum levels. In three patients with a pronounced initial decline of HCV-RNA levels (> 3 log) sequence analyses of NS5A2209-2248 were performed before and after therapy. Compared with the pretreatment amino acid sequence the HCV isolates of these patients revealed more mutations in the NS5A2209-2248 region after therapy. These findings from European patients indicate that the NS5A2209-2248 region of HCV does not represent a common interferon sensitivity determining region.