Surveillance of mosquito populations for virus activity is not often performed by small, vector-control districts because they do not have the financial resources to use virus isolation, or newer methods such as the polymerase chain reaction. Consequently, development and refinements of rapid, sensitive, and simple enzyme-linked immunosorbent assays (ELISAs) applicable to a wide variety of public health settings are justified. We have developed an antigen-capture ELISA for the detection of eastern equine encephalitis (EEE) virus in mosquitoes that uses both monoclonal capture and detector antibodies. The sensitivity of this assay is 4.0-5.0 log10 plaque-forming units/ml, which is comparable to previously published EEE antigen-capture assays developed with polyclonal antibody reagents. This test identifies only North American strains of EEE virus and does not react with either western equine encephalitis or Highlands J viruses. Test sensitivity was enhanced by sonicating mosquito pools, treating them with Triton X-100, and increasing the time and temperature of antigen incubation. The conversion of this ELISA to a monoclonal antibody-based format should result in a readily standardizable and transferable assay that will permit laboratories lacking virus isolation facilities to conduct EEE virus surveillance.