Sera from 360 ciconiform and pelecaniform birds collected in Florida (USA) from 1974 to 1990 were tested for serum neutralizing (SN) antibodies to eastern equine encephalitis (EEE), St. Louis encephalitis (SLE), and Everglades (EVE) viruses. Serum neutralizing antibodies to EEE virus were detected in 2%, to SLE virus in 7%, and to EVE virus in none of the samples. Pelecaniform birds (16%) had a higher antibody prevalence (P < 0.02) for SLE virus than did ciconiform birds (5%). Virus could not be isolated from 67 samples. Nestling birds with SN antibodies to both EEE and SLE viruses were found in both fresh water and marine colonies. Antibodies were more prevalent in adult and fledged juvenile birds than in nestlings.