AbstractSpecific viral laboratory diagnosis of primary Epstein–Barr Virus (EBV) infection is usually based on antibody‐detection assays. During acute, lytic phase of infection, viral DNA can also be detected in serum. In the present study, the diagnostic utility of EBV DNA detection and quantitation in serum in primary EBV infection was investigated. The level of EBV DNA in the serum of 98 immunocompetent patients aged 1–47 years with symptomatic, antibody‐confirmed EBV primary infection was assessed using a quantitative real‐time PCR assay. The association between viral load and time after onset of disease, age and clinical and laboratory data was investigated. Quantitative PCR detected EBV DNA in 93 of 98 samples (94.9%), and the measured viral loads ranged from 3.8 × 101 to 6.6 × 104 copies/ml. EBV DNA detection exhibited a sensitivity of 94.9% and a specificity of 97.4% for primary EBV infection. EBV DNA was always detectable until day 12 after onset of symptoms, whereas no further positive PCR results were found after a period of 22 days after onset of disease. Detection of EBV DNA also showed a clearer association with the clinical manifestation of disease than the presence of EBV specific VCA IgG antibodies of low avidity. EBV DNA load was found to correlate inversely with the time after onset of disease (P < 0.001), and higher viral load levels were detected in younger (P = 0.009) and in hospitalized patients (P = 0.038). The results indicate that real‐time PCR is a reliable tool for diagnosis of primary EBV infection early in the course of disease. In addition, EBV DNA detection may serve as a useful diagnostic supplement in serologically indeterminate EBV infections. J. Med. Virol. 75:54–58, 2005. © 2005 Wiley‐Liss, Inc.