An HHV-6 antigen capture assay measuring gp116/64/54 antigen was developed. This ELISA is specific for HHV-6 Variants A and B, does not cross react with other human herpesviruses, is sensitive, stable, quantitative, and can detect antigen in body fluids and cell cultures. Relative to virus isolation or techniques for measuring HHV-6 nucleic acids, the assay is much simpler and less expensive to perform. Plasmas/sera (413) obtained from healthy donors, children with Exanthem subitum, febrile illnesses, patients with Chronic Fatigue Syndrome, and AIDS patients tested by antigen capture assay demonstrated that the assay is useful in clinical laboratory settings. The capture assay can also be used to monitor cell cultures for virus isolation, production, quantitation, and antiviral agent screening.