Birds are the major amplifying host for West Nile virus (WNV), a flavivirus that may affect humans and transmitted by bloodsucking vectors. Eleonora's Falcons (Falco eleonorae) migrate to the Canary Islands annually from WNV-endemic regions. To investigate the possible role of Eleonora's Falcons in the circulation of WNV, we measured WNV-specific antibodies in 81 falcons captured in 2006. None of the nestlings but 14.8% of the adults had WNV-neutralizing antibodies. RT-PCR did not detect flaviviruses in nonculicine ectoparasites (n=231) of the falcons. These findings suggest that WNV infection did not occur locally, but rather on the wintering grounds or during migration.