West Nile virus (WNV) is a mosquito-borne flavivirus that primarily infects birds but occasionally also infects humans and horses. In recent years, the frequency of WNV outbreaks in humans has increased, and these outbreaks have been associated with a higher incidence of severe disease. In 1999, the geographical distribution of WNV expanded to the Western hemisphere. WNV has a positive strand RNA genome of about 11 kb that encodes a single polyprotein. WNV replicates in the cytoplasm of infected cells. Although there are still many questions to be answered, a large body of data on the molecular biology of WNV and other flaviviruses has already been obtained. Aspects of virion structure, the viral replication cycle, viral protein function, genome structure, conserved viral elements, host factors, virus-host interactions, and vaccines are discussed in this review.