A flavivirus (strain 97-103) was isolated from Culex pipens mosquitoes in 1997 following floods in South Moravia, Czech Republic. The strain exhibited close antigenic relationship to West Nile virus (WNV) prototype strain Eg-101 in a cross-neutralization test. In this study, mouse pathogenicity characteristics and the complete nucleotide and putative amino acid sequences of isolate 97-103, named Rabensburg virus (RabV) after a nearby Austrian city, were determined. RabV shares only 75%-77% nucleotide identity and 89%-90% amino acid identity with representative strains of WNV lineages 1 and 2. Another RabV strain (99-222) was isolated in the same location two years later; it showed >99% nucleotide identity to strain 97-103. Phylogenetic analyses of RabV, WNV strains, and other members of the Japanese encephalitis virus (JEV) complex clearly demonstrated that RabV is either a new (third) lineage of WNV or a novel flavivirus of the JEV group.