Strains of tick-borne encephalitis (TBE) virus isolated from ticks in natural foci in Austria were compared to strains isolated from the same foci 14 years previously. Comparative peptide mapping of the envelope (E) glycoproteins as well as analysis of the antigenic structure of the E proteins by the use of 14 monoclonal antibodies defining different epitopes did not provide evidence for antigenic variation. The same also holds true for isolates from a probably newly established natural focus in Western Austria. These results confirm previous data by showing that under natural ecological conditions TBE virus is quite stable and does not undergo major antigenic changes.