In October 2002, West Nile virus (WNV) was identified in farmed American alligators (Alligator mississippiensis) in Florida showing clinical signs and having microscopic lesions indicative of central nervous system disease. To perform seroepidemiologic studies, an indirect enzyme-linked immunosorbent assay (ELISA) was developed to determine exposure of captive and wild alligators to WNV. To validate the test, a group of WNV-seropositive and -seronegative alligators were identified at the affected farm using hemagglutination inhibition (HAI) and the plaque reduction neutralization test (PRNT). The indirect ELISA utilized a rabbit anti-alligator immunoglobulins polyclonal antibody as the secondary antibody, and inactivated WNV-infected Vero cells were used as the coating antigen. For all samples (n=58), the results of the ELISA were consistent with the HAI and PRNT findings. Plasma was collected from 669 free-ranging alligators from 21 sites across Florida in April and October 2003. Four samples collected in April and six in October were positive for WNV antibodies using HAI, PRNT, and the indirect ELISA. This indicated that wild alligators in Florida have been exposed to WNV. These findings can be used as a baseline for future surveys.