A population of 2,024 voluntary blood donors was tested for anti-EBNA-1 IgG and anti-VCA IgG serum antibodies to define the EBV infection rate and to compare two different epidemiological markers. Initial screening was performed with a sensitive EBNA-1-IgG ELISA based on recombinant antigen (Biotest) and a VCA-IgG ELISA based on conventional antigen. Both ELISAs had concordant results in 90.4% of the sera. The infection rate was found to be 96.8%. The expected immune status VCA+/EBNA+ was observed in 98.1% of the seropositives. The comparison of sensitivity, specificity, and predictive values between the two screening assays underlines the superiority of the EBNA-1 ELISA. The marker anti-EBNA-1 IgG as detected by a sensitive ELISA (Biotest anti-EBV recombinant) is suitable for defining previous EBV infection (positive predictive value 99.8%). The high infection rate in the adult population, however, renders the supply with EBV-negative blood rather difficult.