Little is known about hepatitis C virus (HCV) breakthrough during antiviral therapy, although it would help in understanding HCV resistance to current antiviral treatments. To analyse the implication of virological factors and the vigour of humoral immune responses in this phenomenon, we studied nine chronic hepatitis C patients with a viral breakthrough during IFN/ribavirin combination therapy, as well as five responders and five non-responders. The IRES and regions coding for the capsid protein, the PePHD domain of envelope glycoprotein E2 and the NS5A and 5B proteins were amplified by RT-PCR before treatment, before and during breakthrough, and after treatment. The major variant sequence was obtained by direct sequencing. The heterogeneity of quasispecies was studied by SSCP in all patients and sequencing after cloning in seven genotype 1b-infected patients. Humoral responses against HCV epitopes were also analysed. The major sequences of IRES, PePHD, and NS5B remained stable during treatment, regardless of the treatment response. However, the capsid protein and the regions flanking PePHD showed sequence variations in breakthrough patients, although no specific mutation was identified. The variable V3 region of NS5A, but not the PKR-binding domain and the ISDR, seemed to be associated with differences in response to treatment. The analysis of HCV quasispecies revealed no characteristic pattern during treatment in breakthrough patients, whose HCV genome profiles looked most similar to that of non-responders. The humoral response was similar between groups. In conclusion, viral breakthrough does not seem to be due to selection of resistant strains with signature mutations. J. Med. Virol. 74:41–53, 2004. © 2004 Wiley-Liss, Inc.