High titers of virus-neutralizing antibodies directed against glycoprotein gC of Pseudorabies virus (PRV) (Suid herpesvirus 1) are generally observed in the serum of immunized pigs. A known function of the glycoprotein gC is to mediate attachment of PRV to target cells through distinct viral heparin-binding domains (HBDs). Therefore, it was suggested that the virus-neutralizing activity of anti-PRV sera is directed against HBDs on gC. To address this issue, sera with high virus-neutralizing activity against gC were used to characterize the anti-gC response. Epitope mapping demonstrated that amino acids of HBDs are part of an antigenic antibody binding domain which is located in the N-terminal part of gC. Binding of antibodies to this antigenic domain of gC was further shown to interfere with the viral attachment. Therefore, these results show that the viral HBDs are accessible targets for the humoral anti-PRV response even after tolerance induction against self-proteins, which utilize similar HBDs to promote host protein-protein interactions. The findings indicate that the host"s immune system can specifically block the attachment function of PRV gC. Since HBDs promote the attachment of a number of herpesviruses, the design of future antiherpesvirus vaccines should aim to induce a humoral immune response that prevents HBD-mediated viral attachment.