Eastern gray squirrels (Sciurus carolinensis) have shown high West Nile virus (WNV) seroprevalence, and WNV infection has been suggested as a cause of morbidity and mortality in this species. We experimentally infected nine eastern gray squirrels with WNV to determine the clinical effects of infection and to assess their potential role as amplifying hosts. We observed no morbidity or mortality attributable to WNV infection, but lesions were apparent in several organs. We detected mean viremias of 10(5.1) and 10(4.8) plaque-forming units (PFU)/mL on days 3 and 4 post-infection (DPI) and estimated that approximately 2.1% of Culex pipiens feeding on squirrels during 1-5 DPI would become infectious. Thus, S. carolinensis are unlikely to be important amplifying hosts and may instead dampen the intensity of transmission in most host communities. The low viremias and lack of mortality observed in S. carolinensis suggest that they may be useful as sentinels of spillover from the enzootic amplification cycle.