Interferon ␣ (IFN ␣) can suppress the replication of hepatitis C virus (HCV) in chronically infected patients. However, HCV persists in a significant number of patients despite the normalization of alanine transaminase (ALT) during IFN ␣ therapy. In this study, HCV variants in patients under IFN ␣ therapy were characterized to examine their role in viral persistence during the therapy. Sixteen patients selected for this study were infected with HCV genotype 1b and remained HCV RNA positive for at least 1 month after onset of therapy. Nine patients responded to the therapy in terms of normalization of ALT (responders), whereas seven patients did not show a significant decrease of ALT level (nonreseponders). To examine HCV populations in these patients, the HCV 5Ј noncoding region (5Ј NCR) was analyzed by polymerase chain reaction amplification and sequencing. Newly emerging variants of the HCV 5Ј NCR replaced predominant variants present prior to IFN ␣ therapy in six of nine responders. Most predominant HCV variants during IFN ␣ therapy carried a nucleotide substitution G to A at nt 231 within the 5Ј NCR. An analysis of the HCV quasispecies population in one responder revealed that a preexisting variant became predominant under IFN ␣ therapy. These results emphasized the importance of the genetic heterogeneity of the HCV genome for viral resistance to IFN ␣. Five of seven HCV isolates from nonresponders were identical to those found in responders with regard to the nucleotide sequence of the 5Ј NCR. However, no selection of variants of the HCV 5Ј NCR occurred in nonresponders during the course of therapy. We conclude that IFN ␣ treatment leads to the selection of variants of the HCV 5Ј NCR only in responders and may act differently in nonresponders. Our results suggest that the HCV 5Ј NCR may be a target of anti-HCV actions of IFN ␣.