Peripheral blood lymphocytes from 29 patients with autoimmune diseases and eight normal controls were cultured in vitro. Several weeks later, lymphocyte proliferation was observed in many of these cultures. Cell lines were obtained from about half of the donors, at a frequency of approximately 1 in 10(7) lymphocytes. Outgrowth occurred only in cultures from patients who had antibodies to the viral capsid antigen of Epstein-Barr virus (EBV). The proliferating cells also contained EBV nuclear antigen, an occurrence confirming that proliferation was a result of latent EBV infection of the donors. Supernatants from these cell lines were tested for autoantibodies by screening against normal tissues. About 30% of the cell lines made autoantibodies, many of which reacted with smooth muscle or epithelial cells. Because EBV is harbored in a latent form in the majority of the adult population, reactivation of latent EBV infection in vivo may explain the production of autoantibodies seen in a variety of immunoregulatory disorders.