A clinical study was carried out to compare the response rate of two groups of non-responder (NR) hepatitis C virus (HCV) genotype 1 chronically infected patients treated with interferon and ribavirin, with or without amantadine. The viral load decreased more markedly in the group treated by tritherapy including amantadine, but the response rate at the end of treatment was not significantly different between bitherapy and tritherapy. As amantadine could have an antiviral effect on the ion channel activity of the p7 HCV protein, the p7 quasispecies was characterized by cloning and sequencing. Sequence data were analyzed to determine the pattern and significance of p7 genetic heterogeneity and a possible relationship with therapy. Subtype differences were confirmed between p7 HCV genotypes 1a and 1b, and quasispecies analysis showed a reduction of genetic diversity in subtype 1a, but not 1b, during tritherapy. However, the absence of changes at numerous positions, as well as the conservative changes at other positions, indicated the high conservation of the p7 structure. Residue His-17, proposed to interact with amantadine, was fully conserved in both subtypes 1a and 1b, independently of amantadine administration. In conclusion, although the analysis of the p7 sequences revealed a selective pressure during therapy, no specific residues appeared to be linked to the effect of amantadine on viral decline. These results suggest that the potential antiviral effect of amantadine might be non-specific and related to a reduction in endosomal acidification and therefore reduced viral entry of HCV via its pH-dependent pathway.