To investigate if tick-borne encephalitis virus (TBEV) is present in mammalian wildlife species or ticks in The Netherlands, serum samples and ticks were tested for TBEV antibodies and TBEV RNA, respectively. Serum samples were collected from wild boar (666), deer (13), fox (399), and rodents (90), and were tested for TBEV antibodies, using ELISA, and SN test or HI test. Over a period of 4 years, a total of 906 ticks was collected from seven regions in The Netherlands. In four different regions, this was done on a monthly basis and during four consecutive summers. All ticks were tested for TBEV RNA by RT-PCR. TBEV antibody was detected by ELISA in two (0.5%) sera of foxes and 49 (7%) sera of wild boar, but not confirmed by HI or SNT. TBEV RNA was not detected in any of 906 ticks. It was concluded that there is no real evidence for a TBEV reservoir in ticks or wildlife in The Netherlands.