Epstein-Barr virus (EBV) is detected in Hodgkin and Reed-Sternberg (HRS) cells in up to 50% of patients with Hodgkin's disease (HD). HD patients have been reported to express high serum titers against EBV antigens, even prior to the diagnosis of HD. Patients with high serum titers have a poorer prognosis. The aim of this study was to examine the relationship between the presence of EBV in HRS cells and the antibody titers reactive with different EBV antigens. Frozen serum and histopathological tissues were available from 107 untreated HD patients diagnosed between 1979 and 1991. The presence of EBV in the HRS cells was evaluated with immunohistochemistry directed against the LMP-1 antigen and/or with in situ hybridization of EBER-1. Analyses were performed of serum titers against early antigen (EA), diffuse (IgA and IgG) and restricted (IgG), virus-capsid antigen (VCA) (IgA and IgG), and EBV-encoded nuclear antigens (EBNA, EBNA 1, EBNA 2A, EBNA 2B, EBNA 6). EBV was detected in 27/107 (25%) tumor specimens, with a higher proportion in the MC group 8/13 (62%) (p < 0.01). IgG VCA and EBNA were detected in 99/107 (93%), evidence of a previous EBV infection. There were no significant relationships between antibody titers reactive with different EBV antigens and detectable EBV in HRS cells. Furthermore, there did not appear to be any relationship between EBV serology or the presence of EBV in HRS cells and clinical outcome. The role of EBV in the development of HD, especially its relationship to the immunological response, remains unclear.