A lack of antiviral response in patients with chronic hepatitis C treated with pegylated (PEG)-interferon (IFN)-α-2a + ribavirin (RIBA) may be explained by neutralizing antibodies to IFN-α-2a. The aim of this study was to assess neutralizing antibodies to IFN-α-2a and IFN levels in non-responder patients who were re-treated by PEG IFN-α-2a and RIBA for 12 weeks. Non-responders to a first-line treatment of PEG IFN-α-2a + RIBA were included for treatment with PEG IFN-α-2a (180 µg/week) + RIBA (1,000 mg/day if <75 kg, 1,200 mg otherwise) for 48 weeks. HCV RNA was measured at week 12. IFN levels and neutralizing antibodies to IFN-α-2a were measured retrospectively on stored sera at baseline and weeks 4 and 12, using a quantitative sandwich ELISA for neutralizing antibodies to IFN-α-2a. Twenty-three patients were non-responders and 19 patients were responders at week 12 of the initial phase of the second-line treatment. Non-responders and responders did not differ statistically: baseline age (median age 47 vs. 50 years), HCV RNA (median 6.8 vs. 6.4 log(10) copies/ml), gender (70% vs. 73% males), genotype (genotype 1: 91% vs. 80%). The median IFN-α-2a levels (pg/ml) at weeks 0, 4, and 12 (interquartile range) did not differ between the 19 responders to initial phase of second-line treatment and the 23 non-responders: <3.3 (<3.3-371.4), 1457.3 (106.8-3284.8), and 1,652 (90.8-5,000); 84.5 (3.3-277.4), 1407.4 (120.2-2443.4), and 1620.1 (120.2-2287.1), respectively. Among non-selected consecutive non-responder patients, re-treatment with PEG IFN-α-2a + RIBA is associated with virological response regardless of the presence of antibody-mediated resistance to conventional IFN treatment.