Three commercially available enzyme-linked immunosorbent assays (ELISAs) from Gull, Biotest, and Behring (Enzygnost) and two latex agglutination tests for heterophile antibodies (Monolatex [Biotest] and Mono-Lex [Trinity Laboratories]) were evaluated for the diagnosis of primary Epstein-Barr virus (EBV) infection and EBV seropositivity. Two hundred fourteen consecutive samples from 197 patients with symptoms of primary EBV infection were analyzed by the five assays at a clinical microbiology laboratory. The samples were also analyzed independently by immunofluorescence methods at a reference laboratory. According to the reference methods, 37 patients (40 serum samples) had primary EBV infections, 120 patients (127 serum samples) had had past EBV infections, 33 patients (36 serum samples) were seronegative, and 7 patients (11 serum samples) exhibited atypical reactions. The respective sensitivities and specificities for the diagnosis of primary EBV infection were 95 and 100% for the Gull assays, 100 and 94% for the Biotest assays, and 100 and 89%, for the Enzygnost assays. The Monolatex and Mono-Lex methods showed similar sensitivities and specificities (78 to 85% and 100 to 99%, respectively) for the diagnosis of primary EBV infection. This study demonstrates the usefulness of commercially available assays for the rapid diagnosis of primary EBV infection, but also the importance of large-scale testing of routine samples before choosing an assay.