BACKGROUND Epstein-Barr virus (EBV) has been associated with Hodgkin disease (HD), but its relationship to the age of the patients and the histologic subtype is not well defined. The possibility that other lymphotropic herpesviruses, such as cytomegalovirus (CMV) and human herpesvirus type 6 (HHV-6) could be involved in the pathogenesis of HD is also unclear. METHODS Paraffin-embedded material from 77 cases of HD was studied. This consisted of 24 specimens from children (mean age, 11.4 years), 36 from young adults (mean age, 28.8 years), and 17 from older adults (mean age, 53.8 years). EBV was detected in Hodgkin and Reed-Sternberg cells (HR-S) by a sensitive in situ hybridization technique based on the detection of Epstein-Barr-encoded RNA. Viral activity was subsequently demonstrated in HR-S cells by using monoclonal antibodies to EBV latent membrane protein. RESULTS EBV was found in a total of 25 of 77 cases (32%). There was no significant difference in EBV positivity between the different age groups. However, the prevalence of EBV varied between different subtypes: 68% of mixed cellularity cases were found to be positive by contrast with 24% nodular sclerosis, 0% lymphocyte predominant, and 14% lymphocyte depleted. Analysis of the histologic reports of a further 783 cases of HD to determine the age distribution of the different subtypes revealed an absence of an older adult second peak in the age distribution curve. In situ hybridization for CMV and HHV-6 was negative in all cases. CONCLUSIONS It was concluded that EBV is predominantly associated with mixed cellularity HD, and there is no correlation with the age of the patient.