Fox squirrels (Sciurus niger) (five of eight) were infected with West Nile virus (WNV) when challenged by the oral route with 10(2.3) or 10(3.4) plaque forming units (PFU). The mean maximum serum WNV titer of infected fox squirrels was 10(5.1) PFU/mL and ranged from 10(4.6) to 10(5.6) PFU/mL. These levels of viremia are infectious for several mosquito vectors of WNV. This virus was also isolated from swabs of the oral and rectal cavities, and urine swabs between day 5 and 9 postexposure (p.e.) in amounts as high as 10(2.0), 10(2.8), and 10(2) PFU, respectively. WNV RNA was detected in salivary gland and/or kidney tissue of three squirrels between day 65 and 72 p.e. in the presence of WNV neutralizing antibody, suggesting that long-term persistent infection occurs in fox squirrels. These observations justify further studies to determine if nonarthropod transmission and long-term persistent infection occur naturally in fox squirrels and contribute to trans-seasonal maintenance of WNV.