After the 1999 outbreak of West Nile (WN) encephalitis in New York horses, a case definition was developed that specified the clinical signs, coupled with laboratory test results, required to classify cases of WN encephalitis in equines as either probable or confirmed. In 2000, 60 horses from seven states met the criteria for a confirmed case. The cumulative experience from clinical observations and diagnostic testing during the 1999 and 2000 outbreaks of WN encephalitis in horses will contribute to further refinement of diagnostic criteria.