American Kestrels (Falco sparverius) nesting near Hawk Mountain Sanctuary in eastern Pennsylvania exhibited widespread West Nile virus (WNV) seroprevalence in 2004. Here we examine the dynamics of WNV in kestrels nesting near Hawk Mountain Sanctuary in 2004–06. We tested kestrel saliva for viral RNA using oral swabs analyzed by RT-PCR, and all swabs were negative for WNV. We used plaque-reduction neutralization testing (PRNT) to measure WNV antibodies in serum. In 2004, we tested 22 adult kestrels and most (95%) were positive for WNV. In 2005, we tested blood samples from six adults and seven nestlings, and WNV seroprevalence was 33% in adults and 14% in nestlings. In 2006, we sampled seven adults and eight nestlings, and 29% of the adults and no nestlings were positive. WNV was widespread in birds, mosquitoes, and humans in Pennsylvania in 2003, and subsequently has declined in these populations. The prevalence of WNV antibodies in the kestrels was compared to surveillance data for mosquitoes, dead birds, and humans in Pennsylvania. WNV antibody prevalence in kestrels was significantly correlated with prevalence of WNV infection in dead birds from the previous year (correlation coefficient 1.000; P  =  0.009), but not with prevalence in mosquitoes or humans from the previous year or in dead birds, humans, and mosquitoes from the same year. This suggested that the antibody levels observed in the kestrel population we studied resulted from infections that occurred during the previous year. Our data indicated that WNV prevalence has declined in this population of kestrels over time.