Since the initial outbreak of West Nile virus (WNV) in the northeastern United States in 1999, the virus has rapidly spread westward and southward across the USA, causing high mortality in crows as well as sporadic mortality in horses, humans, and a wide variety of birds. In 2002 the epidemic widened as hundreds of equine and human cases and sporadic cases in other mammalian species were reported. This is the first report of WNV infection in three Eastern fox squirrels (Sciurus niger). Neurologic signs included head tilt, uncoordinated movement, paralysis, and tremors. Gross lesions were absent. Microscopic lesions consisted of lymphoplasmacytic inflammation involving the brain, heart, kidney, and liver. Formalin-fixed tissues from the three squirrels were tested for WNV antigen by immunohistochemical staining and for WNV-specific RNA by reverse transcriptase-polymerase chain reaction (RT-PCR). The kidneys of all three squirrels stained positive with immunohistochemistry for WNV, whereas the brain and heart were positive in only one animal. Two of the three squirrels were positive for WNV by RT-PCR.