Tissues obtained from 14 patients with multiple anatomic sites involved by Hodgkin's disease were studied for Epstein-Barr virus (EBV) using in situ hybridization for EBV-encoded RNA (EBER) 1 and immunohistochemical methods for EBV latent membrane protein (LMP) expression. Each patient in this study had two to five separately involved anatomic sites, and all biopsy sites, a total of 43 specimens, were analyzed for EBV. EBV was detected in 6 of 14 (42.8%) patients with Hodgkin's disease, including 5 of 11 (45.4%) with nodular sclerosis and 1 of 3 (33%) with mixed cellularity. In these six patients, all biopsy sites were positive for both EBER1 and LMP. In the EBV-positive cases were analyzed the 3'-end of the EBV LMP1 gene in al sites of disease using polymerase chain reaction. In three patients all sites of disease had a 30-base pair deletion. In two patients, there was discordance between sites of disease, with LMP1 gene deletions in some sites and other sites with the LMP1 gene in the germline configuration. The results of this study demonstrate that EBV, when found in Hodgkin's disease, is detectable in all anatomic sites involved. The presence of the same 30-base pair deletion in the EBV LMP1 gene in all sites of disease in three patients suggests that the deletion occurred before dissemination and that all sites are clonally related. However, the discordance between anatomic sites in two patients suggests that LMP1 gene deletion may also occur as a later event, after dissemination. These results lend further support to the hypothesis that EBV plays a role in the pathogenesis of a subset of cases of Hodgkin's disease.