Only limited data are available on the development of neutralizing antibodies (NAB) in patients with chronic hepatitis C (CHC) treated with pegylated interferon-α (PEG-IFN-α). The aim of this study was to evaluate the immunogenicity of PEG-IFN-α when administered to CHC patients who had or had not previously received standard IFN-α therapy. In addition, the specificities of NAB, together with the ability of leucocyte (LE) -IFN-α to re-establish therapeutic responsiveness in NAB-positive patients, were evaluated. NAB were assessed using a quantitative, standardized, virus-induced cytopathic effect assay. The seroconversion rate to PEG-IFN-α was higher in patients who had received previous standard IFN-α treatment than in those treated exclusively with PEG-IFN-α. Also, NAB produced during PEG-IFN-α therapy were unable to neutralize LE-IFN-α entirely, even though they can neutralize several IFN-α subtypes. In addition, the results indicate that a change to LE-IFN-α therapy can be associated with restoration of clinical responses in NAB-positive patients who had become resistant after showing an initial response to PEG-IFN-α treatment. This study emphasizes the importance of evaluating NAB development in CHC patients who become resistant to PEG-IFN-α treatment, and suggests management alternatives for patients who develop NAB. © 2011 The Authors. Clinical Microbiology and Infection © 2011 European Society of Clinical Microbiology and Infectious Diseases.