The dynamic features of three specific anti-hepatitis C virus (HCV) antibody subpopulations directed against different conformational epitopes of the viral E2 protein (HCV/E2) have been evaluated in patients with primary and persistent HCV infection; the three subpopulations are present in patients infected with different HCV genotypes and have shown a different activity using a pseudovirus neutralization assay (antibodies e301 and e137 exhibiting high neutralizing activity, while antibody e509 enhancement of HCV infectivity). In sequential samples from five patients with primary HCV infection and different virological outcome, all samples tested negative with the single exception of the e509 antibody in a patient not clearing the virus. In sequential samples from 28 patients with persistent infection under treatment with pegylated interferon-alpha plus ribavirin (14 sustained virological responders and 14 non-responders), the therapy did not selectively influence titers of the two neutralizing antibody subpopulations; otherwise, a net increase of the e509 antibody subpopulation related to enhancement of HCV infectivity was observed in non-responders, but not in sustained virological responders (P = 0.0156). This increase was not related to the trend of total anti-HCV/E2 response. The data indicate that a specific antibody response against these epitopes is elicited only late during the infection, thus not influencing virus clearance during primary infection, and that a selective increase of the antibody subpopulation enhancing virus infectivity is observed only in the cohort of patients not responding to antiviral therapy.