An enzyme-linked immunosorbent assay (ELISA) was developed for the major Epstein-Barr virus (EBV) associated antigens. The ELISA assay was based on purified protein components of virus-capsid antigen (VCA), early antigen (EA), membrane antigen (MA) and the nuclear antigen (EBNA). These antigens, except EBNA, were purified on immunoaffinity columns containing monoclonal antibodies, while EBNA was purified by biochemical methods. Screening of 69 human sera including 26 sera from individuals with infectious mononucleosis against these antigens in ELISA showed a good correlation with immunofluorescence titers against the major groups of EBV associated antigens. The results indicated that the ELISA technique using purified EBV proteins could be useful for detecting and monitoring antibody responses to different EBV proteins in patients with EBV-associated diseases.