Diagnosis of acute primary Epstein-Barr Virus (EBV) infection is predominantly performed by serology. Detection of specific antibodies to defined EBV antigens is considered state of the art. Antibodies to EBNA-1 are not produced early in primary infection and a positive EBNA-1 serology is a sign of past infection. Therefore EBNA-1 serology plays a crucial role for EBV routine diagnosis. In the present study the quantitative EBV EBNA-1-IgG-ELISA PKS medac was evaluated regarding its suitability for routine diagnosis. Using clinically and diagnostically defined serum samples (141 from seronegative, 111 from acute infected, and 52 from individuals with past infection) as well as 100 sera from healthy blood donors the diagnostic performance of the assay was investigated. Furthermore, precision, performance of the single-point quantitation (SPQ), and suitability of the assay for automation were evaluated. Compared to the pre-definition of the serum panel a sensitivity of 100% and a specificity of 99.6% was found. The measurement of the blood donor sera resulted in an anti-EBNA-1 IgG prevalence of 93% and an agreement of 99% with the results of a commercial ELISA used as reference. Regarding intra-assay variation, interassay variation (performed manually and automatically), and person-to-person variation a coefficient of variation < 10% was found with reactive samples. A good dilution linearity (r2 = 0.961), an excellent correlation of SPQ vs. the calibration curve (r2 = 0.997), and between the results of manually vs. automatically performed test runs (r2 > 0.995) was found. The evaluation has shown that the assay meets the demands of routine diagnosis very well.