West Nile virus (WNV) is a bird flavivirus capable of infecting horses and humans that is transmitted by blood-sucking vectors. In Europe and Africa, sporadic infections and outbreaks causing human illness and deaths have occurred and have led to 2 mutually nonexclusive hypotheses regarding the circulation of WNV in Europe: (1) the occurrence of endemic sylvatic cycles that occasionally result in human or equine infection, or (2) sporadic seeding of WNV by migratory birds from areas where the virus is endemic in Africa or elsewhere that cause local epizootic foci and eventually lead to infection in humans. To investigate these 2 possibilities, we used a micro virus-neutralization test to examine the prevalence of WNV neutralizing antibodies in 574 individuals belonging to 25 species of birds captured in spring 2004 in Seville (southern Spain). Trans-Saharan migrant species had both higher prevalences and antibody titers than resident and short-distance migrants. This result suggests that trans-Saharan migrants spend part of their life cycles in areas with greater circulation of WNV, or a closely related flavivirus, before their arrival in Spain. On the other hand, seroprevalences assessed in resident birds suggest a low level of WNV circulation in the studied locality. Aside from the question of local circulation, it thus seems that the risk for introduction of strains of WNV from Africa by migratory birds merits further field and experimental studies in Spain.