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To investigate the role of migratory birds in the dissemination of West Nile virus (WNV), we measured the prevalence of infectious WNV and specific WNV neutralizing antibodies in birds, principally Passeriformes, during spring and fall migrations in the Atlantic and Mississippi flyways from 2001–2003. Blood samples were obtained from 13,403 birds, representing 133 species. Specific WNV neutralizing antibody was detected in 254 resident and migratory birds, representing 39 species, and was most commonly detected in northern cardinals (Cardinalis cardinalis ) (9.8%, N = 762) and gray catbirds ( Dumetella carolinensis ) (3.2%, N = 3188). West Nile virus viremias were detected in 19 birds, including 8 gray catbirds, and only during the fall migratory period. These results provide additional evidence that migratory birds may have been a principal agent for the spread of WNV in North America and provide data on the occurrence of WNV in a variety of bird species.