Recent studies performed in Japan have suggested that hepatitis C virus (HCV) genome heterogeneity might be taken as a predictive virological parameter of response to interferon alfa (IFN-α) treatment. However, there is presently no information on the impact of this virological parameter in patients from Western countries infected by different HCV genotypes. We have investigated this issue by using amplification of HCV E2 hypervariable region 1, followed by single-strand conformation polymorphism assay (PCR-SSCP). We have studied 95 French patients infected with various genotypes and treated with IFN-α-2b. We analyzed the impact of the following parameters by univariate and multivariate analyses: HCV heterogeneity, HCV genotype, viral load, and liver histology in response to therapy. Age > 40 years (P < .01), viral load > 35 × 105 Eq/mL (P < .01), genotype 1 (P < .01), and a number of SSCP bands > 3 (P < .001) were significantly associated with nonresponse or relapse; cirrhosis was associated with nonresponse. In multivariate analysis, three variables were independently associated with the absence of long-term response: SSCP bands > 3, genotype 1, and viral load > 35 × 105 with odds ratios of 19, 7.5, and 11.8, respectively. Our data establish the major importance of HCV genome heterogeneity in patients infected with both HCV types associated with low (genotype 1b) or high (genotype 3a) response to IFN-α.