BACKGROUND Tick-borne encephalitis virus (TBEV) is one of the most important causes of human viral infections of the central nervous system in Europe. Currently, the diagnosis of TBE is based on the demonstration of specific antibodies in patient's serum, which appear only several weeks after the infection. OBJECTIVE To determine how successfully can viral RNA be detected by RT-PCR in the samples of body fluids of patients with TBE prior to and after the appearance of antibodies. METHODS Serum, whole blood and CSF samples from 34 patients with a serologically confirmed TBE were collected. Samples were tested for the presence of TBEV RNA by using RT-PCR method. RESULTS Viral RNA was detected in all blood and serum samples collected before the development of antibodies. After the appearance of IgM antibodies, the number of positive samples dropped by at least one third. After the development of IgG antibodies, only 3% of serum and 16% of blood samples tested positive for viral RNA. Samples of cerebrospinal fluid were shown to be inappropriate for the molecular diagnosis of TBE using this assay, since only one sample (10%) that was collected in the sero-negative phase of disease was found positive by the PCR assay. CONCLUSIONS RT-PCR is an efficient method for an early detection of TBEV in blood and serum samples collected prior to the appearance of antibodies. This method can be of valuable use for a differential diagnosis of TBEV infection in patients with febrile illness after a tick bite, particularly in regions where more than one tick-transmitted diseases are endemic.