Flaviviruses contain three structural proteins: Capsid, Membrane and Envelope. Protein E covers the virion surface and is the primary target for neutralizing antibodies. Based on their serological cross-reactivity, flaviviruses are subdivided into distinct serocomplexes. TBEV, which is endemic in Central and Eastern Europe and in the Northern parts of Asia, is divided into the Western European, Far Eastern and Siberian subtype and several other human-pathogenic viruses. Based on virus isolates of the Far Eastern and Western European subtype, several vaccines are commercially available. Protection after vaccination is mediated by antibodies against neutralizing epitopes located on protein E. TBEV hybrid viruses were established that combine identical replication competence in cell culture with subtype-specific antibody binding and neutralization and this has for the first time made quantitative investigation by NT analysis possible. Immunization with the Neudoerfl-based FSME Immun Inject induced equivalent neutralization titers against the European-, Far Eastern- and Siberian TBEV subtypes, and somewhat lower, but likely protective, neutralizing antibody titers against OHFV. To further investigate the importance of epitopes located on protein E for the induction of neutralizing antibodies we took advantage of the fact that the E protein amino acid sequence of two European TBE vaccine strains (Neudoerfl and K23) exhibit differences in only 4 amino acid positions. By the use of sera of vaccinees immunized with two different European vaccines we have demonstrated that an antigenic site in the DI/DII hinge region of TBEV E protein plays a critical role for the induction of neutralizing antibodies upon immunization.