West Nile virus (WNV) which is a flavivirus transmitted by mosquitos, may lead to asymptomatic infection, mild febrile illness or encephalitis. Many sporadic cases and major outbreaks of West Nile fever have been reported worldwide, however, WNV infections have not been well documented in Turkey. The aim of the present study was to determine the prevalence of past WNV infections in a population of blood donors. Blood samples were collected from donors with their informed consent. Samples were processed and tested for WNV IgG by enzyme-linked immunosorbent assay (ELISA) (Euroimmun, Germany) according to the manufacturer's guidelines. Demographic data of the donors were recorded. A total of 2821 serum samples were tested. Among them, 28 samples were found to be WNV IgG positive (0.9%) and 41 of them were indeterminate (1.4%). Thus a total of 69 objects were considered to have encountered WNV (2.4%). All of the IgG positive samples (n = 69) and randomly-selected negative samples (n = 60) were re-analysed for the presence of viral RNA by a commercial real-time reverse transcriptase PCR (LightMix (R) Kit West Nile Virus, TIBMolbiol, Germany). West Nile virus RNA was not found in any of the samples. In conclusion, our data have supported the results of other studies indicating the presence of WNV infection in Turkey. Further larger scale studies are necessary to evaluate the possible risks of WNV infections in our country in terms of blood banking.