Summary. Resolution of hepatitis C virus (HCV) infection requires a complex interplay between innate and adaptative immune responses. The role of lymphocyte subpopulations during combined antiviral treatment remains to be defined. This study was conducted to assess the effect of pegylated interferon-alpha2a (pegIFN-α2a) and ribavirin treatment on peripheral blood lymphocytes, mainly on CD81 expression on B cells and CD4+CD25+CD127low/−FoxP3+ regulatory T cells (Tregs) in patients with chronic HCV infection. Thirty-five patients with chronic HCV infection who started pegIFN-α2a and ribavirin treatment were enrolled. Peripheral blood mononuclear cells (PBMC) were obtained at baseline before treatment (BT), mid-treatment (MT), the end of treatment (ET) and 24 weeks post-treatment (PT). During combined antiviral treatment, a significant decrease in the percentage of CD3+, CD8+, CD3+gamma/delta (γδ)+, CD19+ lymphocyte subpopulations and Tregs was observed. There was also a significant increase in the percentage of the CD4+ lymphocyte subpopulation and in CD81 expression levels on CD19+ B cells when BT was compared with ET (all P < 0.05). Seventeen patients were nonresponders (NR) and 18 had a sustained virological response (SVR). At baseline, NR patients had higher CD81 expression levels on CD19+ B cells (P = 0.017) and a higher Tregs percentage (P = 0.025) than SVR patients. Our results suggest that immunomodulation fluctuates during antiviral treatment and that percentage CD81 expression levels on B cells and Tregs might be useful as an immunological prognostic factor for pegIFN-α2a and ribavirin treatment response in chronic HCV infection.