In September and October 2002, an epizootic of neurologic disease occurred at an alligator farm in Florida (USA). Three affected American alligators (Alligator mississippiensis) were euthanatized and necropsied, and results confirmed infection with West Nile virus (WNV). The most significant microscopic lesions were a moderate heterophilic to lymphoplasmacytic meningoencephalomyelitis, necrotizing hepatitis and splenitis, pancreatic necrosis, myocardial degeneration with necrosis, mild interstitial pneumonia, heterophilic necrotizing stomatitis, and glossitis. Immunohistochemistry identified WNV antigen, with the most intense staining in liver, pancreas, spleen, and brain. Virus isolation and RNA detection by reverse transcription–polymerase chain reaction confirmed WNV infection in plasma and tissue samples. Of the tissues, liver had the highest viral loads (maximum 10^8.9 plaque-forming units [PFU]/0.5cm³), whereas brain and spinal cord had the lowest viral loads (maximum 10^6.6 PFU/0.5cm³ each). Virus titers in plasma ranged from 10^3.6 to 10^6.5 PFU/ml, exceeding the threshold needed to infect Culex quinquefasciatus mosquitoes (10⁵ PFU/ml). Thus, alligators may serve as a vertebrate amplifying host for WNV.