TBEV (Flaviviridae, Flavivirus) is an etiologic agent of tick-borne encephalitis (TBE), the most important arboviral human infection involving the central nervous system. The disease is endemic in a zone extending from central and eastern Europe to Siberia and Japan, and corresponds to the distribution of the ixodid ticks, which act both as the vectors and reservoir of TBEV. Humans acquire infection mainly by the bite of an infected tick. A continuous increase of TBE cases throughout Europe has been observed over a period of 30 years. The objective of this study was a preliminary determination of the infection level of ticks collected in North-Eastern Poland, the endemic area of TBE. Questing Ixodes ricinus ticks (adults, nymphs and larvae) were collected by flagging the lower vegetation in 55 locations in Poland in 2006-2009. A total of 2075 ticks (676 females, 555 males, 799 nymphs and 45 larvae) were collected and examined for the presence of RNA TBEV by nested RT-PCR. The average number of ticks in one pool was 6.98. The minimum infection rate of ticks with TBEV was estimated in total area as 0.96%. TBEV RNA was detected in all of the investigated developmental stages of ticks. The prevalence of viral infection in ticks is a useful indicator of TBE virus circulation and may be used for risk assessment of the degree of natural focus activity and of the risk to contact TBE in a particular natural habitat.