IgG and IgM levels against West Nile virus (WNV) were measured in 20,033 serum samples that were obtained between October 2008 to September 2009 from 9913 blood donors in the district of Ferrara, northeastern Italy. As confirmatory test, a microneutralization assay was used to detect the presence of neutralizing antibodies against WNV. Sixty-eight subjects (0.69%) were positive for anti-WNV by immunofluorescence assay. Large differences in the prevalence of antibodies to WNV were noted between towns in the area evaluated.