Flaviviruses of the Japanese encephalitis virus (JEV) antigenic complex, including West Nile virus (WNV), are recognized as emerging and reemerging pathogens. Circulation of flaviviruses has been recently detected in different mosquito and vertebrate species in several European countries. A serosurvey study was carried out to evaluate the circulation of WNV and other flaviviruses of the JEV antigenic complex in different wild bird species in Spain between 2006 and 2009. Seropositiviy against JEV using a competitive enzyme-linked immunosorbent assay was found in common coot, Montagu's Harrier, black kite, black vulture, Bonelli's eagle, Spanish imperial eagle, Egyptian vulture, and Eurasian spoonbill. Seropositivity to JEV antigenic complex viruses was significantly higher in samples collected during autumn compared with animals sampled during summer. Significantly higher seroprevalence was also observed in 2007 compared with 2009, whereas there were no significant differences in seropositivity among taxonomic levels, migratory versus resident behavior, body size (large vs. medium), or habitats (free-ranging vs. captivity). Neutralizing antibodies against WNV were detected in common coot and Spanish imperial eagle using a virus-neutralization test. Oral shedding of WNV was not detected in any of the Spanish imperial eagles, Egyptian vultures, Eurasian Spoonbills, Lammergeiers, and the Black vultures analyzed by means of real-time reverse transcription-polymerase chain reaction. The results indicate that WNV and others flaviviruses of the JEV antigenic group circulated in migratory and resident wild bird species in Spain between 2007 and 2008. Further studies are necessary to determine the precise role that each of these wild bird species, some of them cataloged as "near threatened," "vulnerable," or "endangered," play in the epidemiology of those viruses. © Copyright 2011, Mary Ann Liebert, Inc.