BACKGROUND A second generation-RSV enzyme immunoassay (EIA) was compared with cell culture and immunofluorescence to determine the improved efficacy of this reformulated system. OBJECTIVE This study was performed to identify whether the EIA might serve an ancillary function during non-operational virology hours, or whether the EIA may serve as a replacement of the commonly used direct fluorescent assay (DFA). METHODS During the 1992-1993 (September-April) RSV season, 124 freshly collected nasopharyngeal (NP) aspirates were tested by the EIA and the DFA for the presence of antigen to the infectious agent. Infectivity was performed by tube culture cytopathic effect (TC-CPE) in parallel with the two antigen detection methodologies. RESULTS Thirteen of the 48 confirmed positive specimens (27%) failed to yield infectious virus by TC-CPE. The sensitivity, specificity, positive and negative predictive values of the EIA were 90, 95, 91 and 94% respectively. This observed sensitivity of 90% using freshly collected NP aspirates, represents a marked improvement over an earlier generation EIA kit. CONCLUSIONS Considering the simplicity and speed of this EIA (10 min), the test is recommended for use by medical personnel when facilities for DFA and traditional virus culture are not readily available.