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West Nile virus lineage 1 (similar to the strains obtained from golden eagles in Spain, 2007) and Usutu virus (similar to the strains obtained from Culex pipiens in Spain, 2006) were detected in pools from Culex perexiguus collected in southern Spain in 2008 and 2009, respectively. This is the first detection and isolation of West Nile virus lineage 1 from mosquitoes in Spain.