From May 1992 through October 1994, sera were collected from 204 domestic emus (Dromaius novaehollandiae) at a ranch in Volusia County, FL, and tested for antibody evidence of arboviral infection. Hemagglutination-inhibition (HI) and neutralizing (NT) antibodies to eastern equine encephalitis (EEE) virus were identified in sera collected during each year. In addition, HI and NT antibodies to St. Louis encephalitis virus were detected in 3 naturally infected emus. Isolations of EEE virus were made from emu blood and tissues collected in 1992 and 1994, when EEE-related mortality in emus was 14% and 1%, respectively. A total of 259 mosquito pools was collected and tested for arbo-viruses during the 3-year study. The EEE virus was isolated from 4 of 140 mosquito pools (2 Anopheles crucians and 2 Culex erraticus pools) in 1992 and 3 of 10 pools (all Culex nigripalpus) in 1994. Emus vaccinated against EEE virus showed evidence of short-term HI antibody acquisition. Evidence of EEE antibody transfer from naturally infected hens to their offspring is reported.