Enzyme immunoassays (EIAs) for eastern equine encephalomyelitis (EEE) and Highlands J (HJ) virus antigens were compared in a retrospective study with standard virus isolation procedures (VIP) for detection of alphavirus-infected mosquito pools. The original VIP was a plaque assay in chick embryo cell culture, and was performed in the years from 1979 to 1981. Using the original VIP as the reference standard, the sensitivity rate of the EIA was 0.7674 and the false negative rate was 0.2326. However, when the storage age and the initial virus titer of the sample were considered, the sensitivity rate increased. For samples containing > 1,500 plaque-forming units (PFU) per ml of virus during the original VIP, the sensitivity rate of the EIA was 0.97; but the rate declined to 0.14 for those originally containing < 500 PFU per ml. Most of the false negatives (68%) occurred with samples containing < 500 PFU per ml. Presumably the low quantitites of virus in these 50 pools were lost during storage and handling; virus was obtained from only 16% (8/50) during reisolation attempts using BHK-21 cells. Specificity of the EIA was excellent; no false positive results were obtained and serological identification was identical to that determined by plaque reduction neutralization in > 98% of the pools examined. Characteristics of the pools, such as pool size, species of mosquitoes, or gravidity did not affect the EIA results. These studies support the use of EIAs in surveillance programs attempting to determine infection rates of known arboviruses in vector populations.