Hybridomas secreting antibodies to the structural glycoprotein of tick-borne encephalitis (TBE) virus were prepared by fusion of X63-Ag8/653 mouse myeloma cells with spleen cells from mice immunized with purified glycoprotein complexes of TBE virus. These antibodies were tested against 10 different TBE virus strains isolated in different European countries over a period of 26 years from different hosts. Quantitative evaluation of enzyme immunoassay results did not reveal any differences in reactivity among these strains, pointing further to the homogeneity of European TBE virus isolates, which has previously been inferred from results obtained by peptide mapping and competitive radioimmunoassay. Hybridomas defining three different antibody-combining sites (epitopes) on the glycoprotein of TBE virus were selected on the basis of cross-reactivity with another flavivirus. West Nile virus, as well as the ability to inhibit hemagglutination. Two epitopes were type specific, and the third was indistinguishably also present on West Nile virus. Hemagglutination was inhibited by monoclonal antibodies reacting with one of the type-specific epitopes as well as the cross-reactive determinant, which is apparently responsible for the broad cross-reactivity among different flaviviruses observed in hemagglutination inhibition tests with polyvalent immune sera.