A clinical study was carried out to evaluate the persistence of tick-borne encephalitis (TBE) antibodies 2 and 3 years after a primary vaccination series (three-dose regimen), and to assess the antibody response to a booster vaccination with FSME-IMMUN. Volunteers (N = 347, 18-67 years) who had received two doses of either FSME-IMMUN or Encepur adults and a third vaccination with FSME-IMMUN were enrolled. Seropositivity rates were assessed by ELISA and neutralization test (NT). After the primary series, seropositivity rates were 99.1% as determined by ELISA and 100% by NT, decreasing to 85% and 96.8% in the first two years and to 88.7% and 95.4% after 3 years. Following booster vaccination, 100% of subjects were seropositive. Age was the only variable with a significant influence on the probability of remaining TBE seropositive 2 or 3 years after the third vaccination. In subjects aged 18-50 years, the pre-booster seropositivity rate was higher (88.7% and 92.3% after 2 and 3 years, respectively) than in those aged 51-67 years (65.5% and 70.9% after 2 and 3 years, respectively). Adverse events after booster vaccination occurred with a low frequency and were predominantly mild. An annual TBE antibody decline rate of 0.58 (based on NT) was estimated to lead to antibody titer decrease from e.g., 260 to 45.6 after 3 years. To conclude, a booster vaccination with FSME-IMMUN, administered 3 years after primary vaccination, is well tolerated and induces a