An enzyme-linked immunosorbent assay (ELISA) was used to measure IgG antibody titers against a synthetic peptide whose sequence was derived from the glycine-alanine repeating region of Epstein-Barr virus nuclear associated antigen 1 (EBNA-1). Antibody titers were determined in sera from 15 normal subjects, sera from 21 normal male siblings of X-linked lymphoproliferative syndrome (XLP) patients, from 20 XLP patients comprising a total of 42 samples, and ten samples before and ten samples after gamma-globulin therapy in ten patients with XLP. Data analysis demonstrated that while there are differences between the ELISA and ACIF, they appear to measure a similar response as demonstrated by their correlation coefficient (0.77) and the GMT to EBNA observed by both methods. No cross-reactivity of cytomegalovirus antibodies to the EBNA-1 peptide was observed by immunoblotting, ELISA or ACIF using adsorption against AD-169 infected MRC-5 cells. However, non-specific binding was observed if samples were not pre-incubated in a 10% goat serum PBS-Tween 20 solution. This pre-treatment removed the non-specific binding that falsely elevated the GMT in approximately 15% of both normal and XLP samples in ELISA. The ELISA system appears to be a sensitive, reproducible and objective test that may be useful for assessing the antibody response of patients to the EBNA-1 protein.