The specific humoral response against polypeptide components of Epstein-Barr virus (EBV), the induced early diffuse antigen (EA-D), in patients with connective tissue diseases, including systemic lupus erythematosus (SLE) and mixed connective tissue disease (MCTD), was investigated by using the immunoblotting technique. The EA(D)-positive sera from patients with infectious mononucleosis (IM), nasopharyngeal carcinoma (NPC), immunocompromised patients (renal transplant recipients and patients with AIDS) as well as the EA(D)-negative sera from patients with Burkitt's lymphoma and from clinically healthy subjects served as controls. Seven major antigenic polypeptides with molecular weights of 33 kDa, 35 kDa, 52 kDa, 54 kDa, 56 kDa, 58 kDa, and 134 kDa were detected reproducibly by the EA(D)-positive reference sera and, in particular, by each of the NPC sera tested. The EA(D)-positive sera from the other groups showed various combinations of detection patterns and few samples reacted with all the major EA(D) polypeptides. Seventy-three percent of sera from SLE and 47% of sera from MCTD were found to react with EA(D). Sixty-one percent of sera from SLE vs. 5% from MCTD detected all the EA(D) polypeptides. These results could either reflect perturbations of the immune response linked to the autoimmune disease or suggest a possible pathogenic role of EBV.