Eastern Equine Encephalitis (EEE) was diagnosed in a flock of 22 African penguins (Spheniscus demersus) in southeastern Connecticut. The outbreak, occurring in late summer, affected 46% of colony birds. Initial diagnosis of EEE was based on history and clinical signs, but later confirmation of the diagnosis employed serology, virus isolation, RT-PCR and histopathologic examination. Changes in birds ranged from alterations in hematologic and serum chemistry parameters with no overt clinical signs (11%), to the majority (89%) having clinical signs of anorexia (89%), behavior changes (89%), depression (89%), regurgitation (89%), ataxia (89%), recumbency (33%), vomiting (22%), seizures (22%), and diarrhea (11%). The typical clinical course for this disease was 12 (+/- 5) days with early anorexia, behavioral changes, and vomiting, changing to regurgitation in ~1-2 days, ataxia in ~3-4 days, seizures in 5 days where applicable and followed by a slow resolution, though 50% of affected birds have maintained a subtle, intermittent ataxia. Hematologic changes consisted of an initial heterophilic leukocytosis and regenerative anemia followed by increases in absolute lymphocyte and monocyte counts. Serum chemistry abnormalities in infected birds included liver enzyme (ALP, ALT, AST, GGT, and LDH) elevations consistent with hepatitis, muscle enzyme (LDH and CK) elevations consistent with muscle damage and intramuscular injections, decreased electrolyte (Na+, Cl-) values believed to be associated with vomiting, hyperglycemia associated with the stress of illness, elevations in globulin, triglycerides, cholesterol, and in one animal, elevated BUN and creatinine. Resolution of hematologic and serum chemistry abnormalities occurred 67 (+/- 24) days post onset of clinical signs. An additional 41% of the colony showed concurrent clinical signs similar, though milder, to those described for EEE; West Nile Virus is strongly suspected to be the cause of illness in these cases due to elevated antibody titers, but viral isolation attempts were unproductive. Treatments for affected birds consisted of supportive therapy including antibiotics and antifungal agents, assist/tube feeding, fluid and electrolyte therapy, and anticonvulsants. All infected birds survived with the exception of one juvenile bird that was euthanized due to the severity of its illness and lack of response to supportive therapy. Significant necropsy findings included distension of the distal colon, a lateral bulging of the left cerebral hemisphere with decreased superficial vascularization and cerebral edema. Histopathologic findings were consistent with encephalitis and EEE virus was confirmed by PCR to be present in the posterior brain. An inability to isolate virus from affected birds several weeks following illness suggests successful viral clearance in recovered penguins. To our knowledge this is the first report of EEE in African penguins. This outbreak occurred simultaneously with isolated outbreaks in equine and ratite animals and a documented human case in the nearby communities. The high morbidity of EEE disease in this colony emphasizes the potential severity of arbovirus infections in penguins, suggests a potential need for vaccination of penguins against EEE in endemic areas, and stresses the importance of mosquito control at zoological institutions in the prevention of all arbovirus infections.