Monoclonal antibodies that are broadly reactive with either influenza A or influenza B viruses were used to develop a 2- to 3-h antigen capture time-resolved fluoroimmunoassay (TR FIA) for detecting influenza viral antigens in both original nasopharyngeal aspirate specimens and in tissue cultures inoculated with nose or throat swab specimens. The lower limit of sensitivity of the assay was about 10 pg of protein as determined with purified influenza A nucleoprotein expressed by recombinant DNA. When the TR FIA was performed with 96 nasopharyngeal aspirate specimens collected during outbreaks of influenza A (H3N2) virus and the results were compared with serodiagnosis results with paired sera, the specificity and sensitivity of TR FIA for the demonstration of influenza A infections were 95 and 85%, respectively. In culture confirmation assays, more than 80% of the swab specimens that grew influenza A or B virus within 7 days could be identified by the TR FIA within 48 h of the inoculation of cells. The results are consistent with those previously reported for respiratory syncytial virus and extend the applicability of monoclonal antibody-based TR FIA for the rapid diagnosis of acute respiratory viral infections.