Objective One of the side effects of interferon-alpha (IFN-α) therapy is interferon-induced thyroiditis (IIT). The role of lymphocyte subpopulations in IIT remains to be defined. The aim of this study was to assess different peripheral blood lymphocyte subpopulations, mainly CD4 +CD25 +CD127low/-FoxP3 + regulatory T cells (Tregs), in patients with chronic hepatitis C virus (HCV) infection who developed IIT. Design, patients and methods From 120 patients with chronic HCV who started antiviral treatment, those who developed IIT (IIT patients) were selected and compared with patients who did not develop IIT (Co-HCV). Peripheral blood mononuclear cells were obtained before treatment (BT), mid-treatment (MT), end of treatment (ET), 24 weeks post-treatment (PT) and at appearance of IIT (TT). Results Eleven patients developed IIT: three Hashimoto's thyroiditis, one Graves'disease, one positive antithyroidal antibodies, one nonautoimmune hypothyroidism and five destructive thyroiditis. During antiviral treatment, an increase in CD8 + and in Tregs was observed in both groups. A decrease in CD3 +, CD19 + and NKT lymphocyte subpopulations was also observed (all P < 0 ·05). However, no changes were observed in the percentage of CD4 +, CD3 +γδ + and iNKT lymphocytes, Th1/Th2 balance and Bcl2 expression on B cells when BT was compared with ET. At the appearance of IIT (TT), IIT patients had a higher Th1 response (CCR5 +CCR7 -) (P < 0 ·01) and a higher Tregs percentage (P < 0 ·05) than Co-HCV. Conclusions Our results point to the immunomodulatory effects of IFN-α on different lymphocyte subpopulations and a possible role of Th1 response and Tregs in patients with HCV who developed IIT. © 2011 Blackwell Publishing Ltd.