A region associated with sensitivity to interferon (IFN) has been identified previously in the nonstructural protein 5A (NS5A) of hepatitis C virus (HCV) genotype 1b. A study was undertaken to determine whether the presence of mutations in the NS5A2209-2248 sequence could serve as a predictor of response to IFN therapy in children and adolescents with chronic HCV-1b infection. Sixteen children (M/F ratio = 11:5; mean age 11.7 years, range 5 to 19 years) with chronic HCV-1b infection who received IFN-alpha for 6 months (total dose: 8 MU/kg) were enrolled in this study. Twelve of the children (75%) had an underlying malignant disease. Pretreatment NS5A gene sequences were detected by reverse transcription-nested polymerase chain reaction (RT-nested PCR). PCR products were subjected to direct sequencing by the dideoxy chain termination method. The amino acid sequences of NS5A2209-2248 were compared with the published NS5A2209-2248 sequences of HCV-J. The NS5A2209-2248 sequences were detected in 10(63%) of the 16 children. Eight patients had the wild-type sequences, with no amino acid changes; and two patients had the intermediate type, with only one amino acid change. Four (25%) of the 16 patients responded completely to IFN therapy. Three of the four patients had the wild-type sequences, while none of the patients with the mutant type had a complete response. Serum HCV RNA levels in children with the wild type did not differ from those in patients with the mutant type. This study shows that there is no significant correlation between response to IFN and mutations in NS5A2209-2248. The amino acid sequences in NS5A2209-2248 in young patients with chronic HCV-1b infection appear to be conserved.