BACKGROUND: Tick-borne encephalitis (TBE) is the most important arbovirus disease in parts of Europe and Asia. Its laboratory diagnosis depends on the detection of specific IgM antibodies which can be impeded by (1) long-time persistence of IgM antibodies after infection, (2) vaccine-induced IgM antibodies, and (3) cross-reactive IgM antibodies from other flavivirus infections. OBJECTIVES: To assess the extent of interference factors in the serodiagnosis of TBE that might lead to the false positive assignment of a recent infection. STUDY DESIGN: We quantified TBE virus-specific IgM and IgG antibodies in sera collected at different time points from cohorts of (1) 61 TBE patients, (2) 131 TBE vaccinees, and (3) 42 patients with recent dengue or West Nile virus infections. RESULTS: All of the TBE patients were IgM- and IgG-positive upon hospitalization and 87% of acute TBE sera had IgM antibody titers of >500 Arbitrary Units (AU). These titers rapidly declined and only 16% of TBE patients had low IgM titers ≥9 months after infection. Vaccine-induced as well as flavivirus cross-reactive IgM antibodies were rarely detectable and of low titer. CONCLUSIONS: Most of the potential problems of TBE serodiagnosis can be resolved by the quantification of IgM antibodies in a single serum sample taken upon hospitalization. High IgM values (>500 AU in our assay) are indicative of a recent infection. Lower IgM values, however, may require the analysis of a follow-up sample and/or a specific neutralization assay to exclude the possibilities of IgM persistence, vaccine-induced IgM antibodies or heterologous flavivirus infections.