Disulfide-bonding contributes to the function and antigenicity of many viral envelope-glycoproteins. We assessed here its significance for the HCV E2 envelope-protein and a counterpart deleted for hypervariable region-1 (HVR1). All 18 Cysteine-residues of the antigens were involved in disulfides. Chemical reduction of up to half of these disulfides was compatible with anti-E2 monoclonal antibody reaction, CD81-receptor binding and viral entry while complete reduction abrogated these properties. Addition of 5,5'-dithiobis-2-nitrobenzoic acid had no effect on viral entry. Thus, E2 function is only weakly dependent on its redox-status and cell-entry does not require redox-catalysts, in contrast to a number of enveloped viruses. As E2 is a major neutralizing-antibody target, we examined the effect of disulfide-bonding on E2 antigenicity. We show that reduction of 3 disulfides, as well as deletion of HVR1, improved antibody binding for half of the patient sera tested while it had no effect on the remainder. Small-scale immunization of mice with reduced E2 antigens greatly improved serum reactivity with reduced forms of E2 when compared to immunization using native E2 while deletion of HVR1 only marginally affected the serum's ability to bind the redox intermediates. Immunization with reduced E2 also showed an improved neutralizing-antibody response suggesting that potential epitopes are masked on the disulfide-bonded antigen and that mild reduction may increase the breadth of the antibody-response. Although E2 function is surprisingly independent of its redox-status, its disulfide-bonds mask antigenic domains. E2 redox manipulation may contribute to improved vaccine design.