Using different forms of the envelope glycoprotein E from tick-borne encephalitis virus we investigated the influence of physical and antigenic structure on the efficacy of vaccination. Different protein E-containing preparations were either derived from purified virions or were produced as recombinant proteins in COS cells. These included soluble dimeric forms (virion-derived protein E dimers with and without membrane anchor; recombinant protein E dimers without membrane anchor), micellar aggregates of protein E (rosettes), and recombinant subviral particles (RSPs). The structural differences between these immunogens were verified by sedimentation analysis, immunoblotting and epitope mapping with a panel of monoclonal antibodies. Specific immunogenicities were determined in mice in comparison to formalin-inactivated whole virus. Rosettes and RSPs were excellent immunogens and exhibited similar efficacies as inactivated virus in terms of antibody induction and protection against challenge, whereas all of the soluble forms were much less immunogenic. These data emphasize the importance of the immunogen's antigenic and physical structure for an effective stimulation of the immune system and indicate that RSPs represent an excellent candidate for a recombinant vaccine against tick-borne encephalitis.