The Epstein–Barr virus (EBV) early antigen (EA) complex consists of multiple proteins with relevance for diagnosis of acute, chronic and malignant EBV related diseases, including nasopharyngeal carcinoma (NPC). In a recent study, it was found that the molecular diversity of EBV‐specific IgG and IgA antibody responses in NPC patients and demonstrated that these reflect independent B‐cell triggering leading to distinct EBV antigen‐recognition profiles. The fine‐specificity of NPC‐related IgG and IgA responses was explored further against defined recombinant and synthetic EBV‐EA antigens using immunofluorescence, immunoblot and ELISA techniques and determined their diagnostic value in a large panel of sera from NPC (n = 154), non‐NPC tumor patients (n = 133), acute mononucleosis patients (n = 70) and healthy EBV carriers (n = 259). Individual recombinant EBV‐EA markers yielded sensitivity/specificity values not exceeding 86%, whereas selected EA‐specific peptide epitopes were rather poorly recognized by IgG and IgA antibodies in NPC sera. Surprisingly, we found that a “low salt” native EA‐protein extract reproducibly prepared from purified nuclei of EA‐induced HH514 cells, and containing characteristic EA(D)‐polypeptides, such as p47‐54 (BMRF1), p138 (BALF2), p55‐DNAse (BGLF5), and p65‐TK (BXLF1), but without viral capsid (VCA) or nuclear antigen (EBNA) reactivity, gave highest sensitivity (90.4%) and specificity (95.5%) values for NPC diagnosis in both IgG and IgA ELISA. The data support further the notion that EBV‐EA reactive IgG and IgA antibodies in NPC patients are directed against distinct conformational and—in part—linear epitopes on EBV‐specific proteins, barely recognized in other EBV‐related syndromes. The use of a defined native EBV EA‐specific antigen opens the way to further improve serological diagnosis of NPC. J. Med. Virol. 79:1710–1721, 2007. © 2007 Wiley‐Liss, Inc.