An enzyme-linked immunosorbent assay (ELISA) that detects IgM antibody to a peptide component of the Epstein-Barr virus (EBV) nuclear antigen (EBNA-1) was compared with a conventional rapid heterophil antibody method for the rapid diagnosis of infectious mononucleosis. Discrepancies between the two methods were further analyzed using an indirect immunofluorescence assay to detect antibodies to EBV antigens. We evaluated 298 cases of suspected infectious mononucleosis. The ELISA was very sensitive (98.7%) and able to detect some cases (seven (9%) of 75 confirmed positives) that were negative by the rapid heterophil antibody test, but confirmed by immunofluorescence. However, ∼17% of all positive tests could not be confirmed by EBV-specific immunofluorescence; thus, the overall positive predictive value was 83%; negative predictive value was 99.5%; and specificity was 93%. The high rate of false-positive tests makes this rapid ELISA unsuitable for the diagnosis of infectious mononucleosis.