Big brown (Eptesicus fuscus) and Mexican free-tailed (Tadarida brasiliensis) bats were inoculated with the New York 99 strain of West Nile virus to assess their potential to serve as amplifying hosts and determine the clinical effect of infection. Groups of three or four bats were bled at daily intervals between 1 and 6 days after inoculation to determine the pattern of viremia. Beginning 2 days after inoculation, virus was isolated each day from one or more E. fuscus bats, in titers ranging from 10 to 180 plaque-forming units per milliliter of serum. Virus was not isolated from any of the sera collected from T. brasiliensis bats. None of the bats from either species showed clinical signs associated with exposure to virus. Sera from an additional 149 bats collected in Louisiana in 2002 during an epizootic of West Nile fever were tested for antibodies to virus, and two were found to be positive. These data suggest that bats from these two widely distributed species are unlikely to serve as amplifying hosts for West Nile virus.