The emergence of West Nile virus (WNV) was expected in Austria since the initial discovery of the infection in neighbouring Hungary in 2003/2004. In 2008 six cases of West Nile disease were diagnosed at the Institute for Veterinary Disease Control Mödling, Austrian Agency for Health and Food Safety (AGES), involving five goshawks (Accipiter gentilis) and one gyrfalcon (Falco rusticolus), which were found dead in the eastern Austrian federal states of Lower Austria, Vienna and Styria, respectively. Pathomorphological and immunohistochemical findings suggested a WNV infection. Virus was isolated in embryonated specific pathogen free chicken eggs and propagated in mouse neuroblastoma cells (NA), in which a cytopathic effect occurred. The virus was identified and characterised by electron microscopic examination and molecular detection using RT-PCR, sequencing, and phylogenetic analysis. The Austrian WNV sequences exhibited nucleotide identities of 99.9% to the lineage 2 WNV sequences described in Hungary since 2004. In addition, 71 sera of 14 different bird species were screened for the presence of WNV antibodies using a commercial ELISA: 43.7% of the tested samples showed antibody titers. Selected positive sera were also subjected to WNV neutralisation tests, in which the ELISA results were verified in 66%. The results of this study confirm unambiguously the presence of a lineage 2 WNV infection in birds of prey in the eastern part of Austria.