Hepatitis C virus (HCV) populations persist in vivo as a mixture of heterogeneous viruses called quasispecies. The relationship between the genetic heterogeneity of these variants and their responses to antiviral treatment remains to be elucidated. We have studied 26 virus strains to determine the influence of hypervariable region 1 (HVR-1) of the HCV genome on the effectiveness of alpha interferon (IFN-alpha) therapy. Following PCR amplification, we cloned and sequenced HVR-1. Pretreatment serum samples from 13 individuals with chronic hepatitis C whose virus was subsequently eradicated by treatment were compared with samples from 13 nonresponders matched according to the major factors known to influence the response, i.e., sex, genotype, and pretreatment serum HCV RNA concentration. The degree of virus variation was assessed by analyzing 20 clones per sample and by calculating nucleotide sequence entropy (complexity) and genetic distances (diversity). Types of mutational changes were also determined by calculating nonsynonymous substitutions per nonsynonymous site (K(a)) and synonymous substitutions per synonymous site (K(s)). The paired-comparison analysis of the nucleotide sequence entropy and genetic distance showed no statistical differences between responders and nonresponders. By contrast, nonsynonymous substitutions were more frequent than synonymous substitutions (P </= 0.05) in responders, but there was no significant difference in nonresponders. Nonsynonymous substitutions tended to be more frequent than synonymous substitutions in women (P = 0.06) but not in men. Nucleotide entropy and genetic distances were significantly related to serum RNA concentration (P </= 0.01). Our findings suggest that after controlling for the major determinants of interferon response, neither complexity nor diversity of the HVR-1 region is associated per se with virus eradication. Because a higher proportion of nonsynonymous substitutions than synonymous substitutions was found only in responders, host anti-HCV-specific immune response rather than viral factors may be playing an important role in the interferon response.