West Nile virus (WNV) has been identified in nearly 300 species of wild birds, including raptors, in North America since its introduction in New York City in 1999. American Kestrels (Falco sparverius) are susceptible to WNV infection, and the numbers of these birds have declined along the Atlantic coast in recent years. We examined the population biology and WNV exposure of kestrels breeding in the area surrounding Hawk Mountain Sanctuary in Kempton, Pennsylvania, USA. The reproductive biology of kestrels in this area was studied from 1992 until 2004. The number of kestrels breeding in nestboxes in 2004 was only 44% of the 6-yr mean observed prior to 1999. During the 2004 nesting season (study period: 8 June through 22 July 2004), adult kestrels were trapped near the site of their nestboxes. Blood samples were obtained, and serum antibodies specific to WNV were quantified using a plaque reduction neutralization test. Of 22 birds tested, 21 exhibited serum antibodies to WNV, suggesting that most (95%) of the adult kestrels in the population had been exposed to WNV.