We investigated mosquito and bird involvement in West Nile virus (WNV) transmission in July 2001 in Jefferson County, FL, and Lowndes County, GA. We detected 16 WNV-infected pools from Culex quinquefasciatus, Cx. salinarius, Cx. nigripalpus, and Culiseta melanura. In Florida, 11% of 353 bird sera neutralized WNV. Antibody prevalence was greatest in northern cardinal (Cardinalis cardinalis, 75%), northern mockingbird (Mimus polyglottus, 50%), common ground-dove (Columbina passerina, 25%), common grackle (Quiscalus quiscula, 15%), domestic chicken (Gallus gallus, 16%), and house sparrow (Passer domesticus, 11%). Antibody-positive birds were detected in nine of 11 locations, among which prevalence in chickens ranged from 0% to 100%. Seropositive chickens were detected in Georgia as well. The primary transmission cycle of WNV in the southeastern United States apparently involves Culex mosquitoes and passerine birds. Chickens are frequently infected and may serve as effective sentinels in this region.