The Enzygnost anti-Epstein-Barr virus enzyme-linked immunosorbent assay (ELISA) system, which is based on a defined antigen mixture and on detection of antibodies of the immunoglobulin G (IgG), IgM, and IgA classes, was evaluated for its reliability in diagnosing Epstein-Barr virus infections in childhood. With samples from 66 children, the Epstein-Barr virus status and the infection phase were defined by indirect immunofluorescence and anticomplement fluorescence assays: 11 children were seronegative, 8 had a primary infection, 20 had a recent primary or past infection, and in 27 a reactivated Epstein-Barr virus infection was diagnosed. When applying the Enzygnost ELISAs, 15 serum samples (22.7%) were not interpretable due to indeterminate results in at least one of the assays used and were therefore excluded from further evaluation. The respective sensitivities and specificities for the diagnosis of seronegativity were 100 and 100%, those for the diagnosis of primary infection were 100 and 97%, those for the diagnosis of recent primary or past infection were 100 and 52%, and those for the diagnosis of reactivated infection were 10 and 100%. This poor performance of the Enzygnost system with reactivated infections is due to the prerequisite of an IgG antibody value of >650 IU/ml for the diagnosis of viral activity, which was fulfilled in only two of the children. Despite the high rate of indeterminate results, the Enzygnost system is useful in diagnosing acute and past Epstein-Barr virus infection in childhood. For serological diagnosis of viral activity in childhood, a supplementary assay is necessary.