A highly sensitive and nonradioactive microplate hybridization assay for the detection of Epstein-Barr virus (EBV)-specific polymerase chain reaction (PCR) product was developed. The PCR product is labelled by adding digoxigenin-dUTP directly to the reaction mixture and, after denaturation, is captured by a microtitre plate coated with an extravidin-linked biotinylated probe. Captured products are reacted with a peroxidase-conjugated anti-digoxigenin antibody and detected using tetramethylbenzidine. The assay detected less than ten EBV genomes in a background EBV-negative DNA of 0.75 microg and, when tested on clinical samples, it was able to define the viral load in throat washings of patients with acute infectious mononucleosis, immunosuppressed patients with HIV infection, and rare normal individuals who shed the virus in the oropharynx.