Sensitivity and specificity of an antigen-capture ELISA vs virus isolation in cell culture were evaluated for the detection of eastern equine encephalomyelitis (EEE) virus in the brain tissue of naturally infected equids. Brain specimens from 16 equids with neurologic disease were examined by ELISA and by inoculation onto baby hamster kidney cell cultures. Of 10 brain samples from which virus was isolated in the cell culture bioassay, all were correctly identified as containing EEE virus antigen by ELISA. None of the remaining 6 specimens, without detectable infectious EEE virus, contained detectable antigen. Sensitivity and specificity of the ELISA were 100% with no false-positive or false-negative results. The antigen-capture ELISA was a rapid, sensitive, specific, and simple alternative to a traditional bioassay for the detection of EEE virus.