A sensitive time-resolved fluoroimmunoassay (TR-FIA), adapted from TR-FIA procedures already described, was developed with monoclonal antibodies and compared with several enzyme immunoassays (EIAs) for detecting adenovirus antigens in clinical specimens. The most sensitive EIA was an all-monoclonal assay with biotin-labeled detector antibody and streptavidin-peroxidase conjugate. All tests were evaluated with nasopharyngeal aspirate specimens from respiratory illness, with tissue homogenates from patients with systemic infection, and with stool specimens from gastrointestinal illnesses. For respiratory and tissue specimens, the TR-FIA detected adenovirus in 85% of the specimens positive by culture, which was a sensitivity similar to those of the all-monoclonal biotin-avidin EIA (79%) and the polyclonal-capture biotin-avidin EIA (88%). For stool specimens, the TR-FIA detected adenovirus in 100% of the specimens positive by culture, which was a decidedly higher sensitivity than either EIA format (78 and 75%, respectively). The TR-FIA was shown to be an efficient, flexible, and specific test for large numbers of clinical specimens.