AIM OF THE STUDY: The analysis of cases of tick-borne encephalitis (TBE) laboratory diagnosed at the Department of Virology of the Institute of Clinical Microbiology of the University Hospital in Hradec Králové in 2003-2006 and comparison of the data from 2003-2006, 1999-2002 and 1995-1998. Another objective was to compare two routinely used ELISA (enzyme-linked immunosorbent assay) diagnostic kits for TBE. MATERIAL AND METHODS: The samples from 3284 patients tested by the Department of Virology of the University Hospital in Hradec Králové in 2003-2006 were analyzed. The detection of the specific IgM and IgG antibodies in serum and cerebrospinal fluid specimens was performed using two ELISA diagnostic kits (Euroimmun, Germany, and Test-Line Ltd, Clinical Diagnostics, Czech Republic). Non-specific reactivity of IgM antibodies was only analyzed in 2004-2006. RESULTS: In 2003-2006, TBE was diagnosed in 163 patients of the University Hospital in Hradec Králové aged from 3.5 months to 80 years. The number was by 48 cases (29%) higher than that in 1995-1998 and by 36 cases (22%) higher than that in 1999-2002. TBE was confirmed in 94 males (57.7%) and 69 females (42.3%). More than half of the patients were aged above 41 years (94/163, i.e., 57.6 %) and 60 patients (37 %) were older than 51 years. A biphasic course of infection was observed in 93/136 cases (68.4%). As many as 101/136 patients (74 %) had a history of a tick bite, one patient reported the consumption of non-pasteurized milk. Thirty-eight (1.4 %) specimens showed non-specific reactivity of IgM antibodies. None of the cases showed cerebrospinal fluid reactivity in the absence of serum reactivity. CONCLUSIONS: Increase in the diagnosed TBE cases was reported in 2003-2006 in the East Bohemia Region. It was due mainly to more severe clinical forms of TBE that were more frequent than the milder ones. The distribution of the confirmed TBE cases remained unchanged in terms of age and sex, but the seasonal peak moved from summer to autumn. ELISA proved suitable for use in the routine diagnosis of TBE. Both of the tested commercial kits are of good quality. Nevertheless, in view of possible non-specific reactivity of IgM antibodies, IgG antibodies need to be tested in both the first acute serum specimen and paired sera. The detection of antibodies in cerebrospinal fluid is unlikely to be of diagnostic benefit for TBE. Close cooperation between the attending physician and clinical microbiologist is crucial.