A one-step time-resolved fluoroimmunoassay (TR-FIA) and a conventional two-step enzyme immunoassay (EIA) for the detection of rubella virus antigen were developed. Two noncompetitive mouse monoclonal antibodies reactive with epitopes on the E l polypeptide of rubella virus served as immunoreagents. One of the monoclones (7A6) was used for coating the solid phase, and the other (2C3) was labeled with either Europium chelate or with horseradish peroxidase. For TR-FIA, the specimen was incubated simultaneously with the label at 4ยฐC overnight. EIA required an overnight incubation with the specimen and after washing another 1 hr of incubation at 37ยฐC with the conjugate. The sensitivity of TR-FIA was 10 pg in an assay volume of 100 ~l , and the sensitivity of EIA was between 50 and 100 pg. Antigens could be detected by TR-FIA in supernatant of cultures of Vero cells 48 hr after inoculation with approximately 1 TCID50, while cytopathogenic effect (CPE) at that time was detected only in cultures inoculated with lo5 TCID50 or more. Virus mixed with human amniotic fluid containing antirubella-specific IgG was detectable after an incubation at 37ยฐC for 5 days. The assays may find applications in prenatal diagnosis of intrauterine rubella infection, in early identification of viral antigens in cell culture and in monitoring production, concentration, and purification of rubella antigen for antibody assays.