West Nile virus (WNV), a member of Flaviviridae family, is an enveloped, icosahedral, single-stranded positive-sense RNA virus. WNV is transmitted to humans by infected mosquitoe (especially Culex spp.) bites and cause a variety of clinical outcomes, ranging from asymptomatic infection to severe meningoencephalitis. The aims of this study were to determine and confirm the WNV seroprevalence in a chosen healthy population and to provide epidemiological data for Turkey about the recent status of the infection at our region. A total of 1200 serum samples collected from blood donors (400 were female, 800 were male; age range: 18-61 years, mean age: 37.8) who were admitted to Hacettepe University Hospital Blood Donation Center between April to December 2009, were included to the study. The presence of WNV IgG antibodies were screened by ELISA (Euroimmun, Germany), and the positive samples have been further investigated by WNV IgG avidity test in order to estimate the time of encounter to the virus. Indirect immunofluorescence antibody (IFA) test (Euroimmun, Germany) and plaque reduction neutralization test (PRNT) which is accepted as the reference method, were performed for the confirmation of WNV IgG positive results. Nineteen (1.6%) of the samples yielded WNV IgG positivity with ELISA, and all of which were IgGs with high avidities (Avidity index values were between 67.8-99.2%). Eight of 19 (42.1%) WNV ELISA IgG positive donors, had risk factors such as joining outdoor activities, contact with mosquitos and ticks and consuming raw milk and milk products. Of 19 samples that were taken into confirmation tests, 15 (78.9%) were found positive with IFA, and 10 (52.6%) were found positive with PRNT. WNV antibody positivity of 10 samples were then confirmed by PRNT, however eight samples which were found positive with both ELISA and IFA yielded negative results with PRNT. This data might indicate that ELISA and IFA methods in which virus-infected cells were used as substrates, have detected non-neutralizing antibodies against viral nucleocapsid antigens rather than the neutralizing antibodies detected against envelope glycoproteins by PRNT method. One sample which yielded low positive result only by ELISA test has been evaluated as a specificity issue of the test. As a result, the positivity rate (19/1200; 1.6%) of WNV IgG detected by ELISA in blood donors, has been confirmed as 0.8% (10/1200) by a gold standard method, PRNT. The data of this study indicated that the prevalence of WNV infections, although low in our region, deserves attention to be considered in surveillance and control programs related to WNV in Turkey.