Recent evidence has shown that Hodgkin's disease (HD) is associated with Epstein-Barr virus (EBV) in a substantial number of cases and that in these cases EBV DNA is localized exclusively to Hodgkin and Reed-Sternberg (RS) cells. The virus genome is not silent in RS cells because two EBV latent gene products, latent membrane protein (LMP) and EB early region (EBER) transcripts, have recently been reported to be expressed in RS cells. However, little information is available about the possible activation of EBV replicative genes in HD. This prompted us to investigate HD biopsies from 96 patients for expression of replicative gene products. Cryostat sections were immunostained with monoclonal antibodies to protein BZLF1, which controls the switch between EBV latency and replication, and also to LMP. LMP was demonstrated in RS cells in 47 cases (49%). Three of the LMP-positive cases (6%), but none of the LMP-negative cases, expressed the BZLF1 protein. BZLF1 positively was confined to rare RS cells. These three cases showed no detectable early, virus capsid, or membrane antigens. Our findings show that activation of EBV immediate early genes occurs only infrequently in RS cells, indicating that control of viral latency is not severely impaired in HD patients.