Abstract Background: Epstein–Barr virus (EBV) infection is associated with infectious mononucleosis, Burkitt's lymphoma, and nasopharyngeal carcinoma. Serologic diagnosis of acute EBV infections has been the method of choice, and tests are available as indirect fluorescent antibody (IFA)- and ELISA-based assays. Objective: To evaluate the ELISA-based EBV assay from Wampole Laboratories (Cranbury, NJ). Methods: One hundred fifty-two consecutive samples received for comprehensive EBV serology were analyzed. Results: A comparison of the Wampole Laboratories' ELISA system with the Gull/Meridian Diagnostics (Cincinnati, OH) IFA, and ELISA assays showed 88% concordance for anti-viral capsid antigen (VCA) IgM ( n=177); 79% concordance for anti-VCA IgG ( n=177); 87% concordance for anti-NA IgG ( n=172); and 48% concordance for anti-EA IgG ( n=165). Using the results from all four antibody assays to identify patients with acute infection, the Wampole system had a 67% concordance with the Gull/Meridian ( n=164). Conclusions: Differences in the specificity of the anti-EA IgG assays (i.e. reactivity against the D component of early antigen (EA-D) (Wampole) vs. reactivity to EA-D and the R component of early antigen (EA-R) (Gull/Meridian)) may have lead to poor concordance (48%) for this particular assay. Because the Wampole system had a <70% overall concordance with the Gull/Meridian system in diagnosis of acute infection, these data suggest that further studies are needed to determine the true clinical sensitivity and specificity of this system.