Hepatitis C virus (HCV) is an RNA virus with the NS5B gene encoding an RNA-dependent RNA polymerase. Interferon-alpha (IFN-alpha) is effective against HCV and its effect is believed to be related to its antiviral activity. To determine whether sequence variations of the HCV NS5B region correlate with response to IFN therapy, pretreatment serum samples from 40 patients with chronic HCV infection who were subsequently treated with IFN (> or = 3 MU thrice weekly for 24 weeks) and had well-characterized biochemical responses were studied. Reverse transcription-polymerase chain reaction (RT-PCR) was performed to generate an approximately 365-bp fragment from which nucleotide sequence and genotypes were determined. By comparing the nucleotide sequences and the encoded amino acid sequences of samples from each group, no response group-specific nucleotide or encoded amino acid substitution was identified. Most of the substitutions identified were synonymous (usually by changes at the third position of the codon). These data suggest that these substitutions were selective rather than spontaneous events. Of the few non-synonymous substitutions identified, none was correlated with subsequent response to IFN, either within or across genotypes.