Anti-hepatitis B virus (HBV) surface-antigen immunoglobulins prepared from human sera are clinical reagents which have been approved for prophylactic treatment in HBV-exposed persons. The passive immunoprophylaxis with immunoglobulins is meant to cross-link viral particles, which are then further cleared by the host's own immune system. While antibodies specific for both anti-S- and anti-preS proteins have been proved to serve as effective anti-viral agents, so far the fine antigen specificity of clinical immunoglobulin preparations has not been determined. Using recombinant proteins covering the hepatitis B surface antigen, in the present study, the specificity of a commercially available immunoglobulin preparation was determined and immunodominant epitopes were mapped. Here, it is shown that the major reactivity of anti-HBV immunoglobulins is directed against the S-protein, and that no reactivity to the preS2 but a weak binding activity to the preS1 region was detectable. The antigen reactivity within the preS1 region was biased to the C-terminal region, which indicates the presence of a putative B-cell epitope. The evaluation of the antigen specificity and determination of novel protective epitopes will provide valuable information for the further development and improvement of prophylactic HBV immunoglobulins.