Summary Hepatitis C virus (HCV) variability affects viral–host interactions. We analysed HCV 5′untranslated region (5′UTR) in sera and peripheral blood mononuclear cells (PBMC) from chronic hepatitis C patients undergoing antiviral treatment. We studied 139 patients treated with pegylated interferon and ribavirin. The primary endpoint was a sustained virological response (SVR) defined as negative HCV RNA level 24 weeks after the end of therapy. 5′UTR was analysed by single-strand conformational polymorphism (SSCP) and sequencing. The pretreatment SSCP pattern in serum and PBMC differed in 26 (18.7%) patients. During therapy, the SSCP pattern remained stable in 65 (60.8%) patients, number of bands declined in 16 (15.0%), and in 18 (16.8%) patients, changes were qualified as ‘shift’ indicating change in band positions. In univariate analysis, there was a significant (P ≤ 0.05) positive association between SVR and pretreatment serum and PBMC dissimilarities, initial viral load