West Nile virus (WNV) is an arbovirus usually transmitted by mosquitoes. The main reservoirs are birds, although the virus may infect several vertebrate species, such as horses and humans. Up to 80% of human infections are asymptomatic. The most frequent clinical presentation is febrile illness, and neuroinvasive disease can occur in less than 1% of cases. Spain is considered a high-risk area for the emergence of WNV due to its climate and the passage of migratory birds from Africa (where the virus is endemic). These birds nest surrounding wetlands where populations of possible vectors for the virus are abundant. Diagnosis of human neurological infections can be made by detection of IgM in serum and/or cerebrospinal fluid samples, demonstration of a four-fold increase in IgG antibodies between acute-phase and convalescent-phase serum samples, or by detection of viral genome by reverse transcription-polymerase chain reaction (especially useful in transplant recipients). Since WNV is a biosafety level 3 agent, techniques that involve cell culture are restricted to laboratories with this level of biosafety, such as reference laboratories. The National Program for the Surveillance of WNV Encephalitis allows the detection of virus circulation among birds and vectors in areas especially favorable for the virus, such as wetlands, and provides information for evaluation of the risk of disease in horses and humans.