The extreme 5′-proximal sequences of the hepatitis C virus (HCV) genome including the 5′untranslated region (5′UTR) and the first 30 nucleotides of the core region are highly conserved, and serve as an internal ribosome entry site (IRES) that initiates the cap-independent translation of HCV polyprotein. Mutations in the IRES sequence have been shown to cause changes in the efficiency of protein translation in vitro. However, the significance of genetic variations in the IRES is not fully known in clinical settings. Pretreatment sera of 25 patients with HCV-1b infection who were treated with interferon were amplified by polymerase chain reaction (PCR), and the IRES sequence was directly sequenced. Correlation of interferon responses or other clinical features with IRES sequence variability was studied. Eleven of 25 patients were sustained responders (SR) of interferon treatment (negative serum HCV RNA and normal alanine transaminase levels for 6 months after the end of interferon treatment), and the other 14 patients were nonresponders ([NR], defined as any patient with positive serum HCV RNA within 6 months after the end of interferon therapy). In each patient, one to four nucleotide substitutions were found compared with the consensus sequence of HCV-1b genotype. There were no differences in the number of nucleotide substitutions between either SR and NR (mean, 1.8 in SR, 2.1 in NR; P = .30), and no specific variations associated with SR or NR were observed. Although NR had significantly higher serum levels of pretreatment HCV RNA than SR (median, 16 vs. <0.5 Meq/mL; P = .02), there was no correlation between the HCV-RNA level and the number of nucleotide substitutions in the IRES (mean, 1.9 nucleotide substitutions in 12 patients with HCV RNA <0.5 Meq/ mL vs. 2.1 nucleotide substitutions in 13 patients with HCV RNA >0.5 Meq/mL; P = .61). Sequence variability of the IRES has no influence on interferon efficacy or serum HCV-RNA concentrations in patients with chronic HCV-1b infection.