Distribution and phenotype of Epstein-Barr virus (EBV)-harboring cells were determined in Hodgkin's disease (HD) biopsies by in situ hybridization with [35S]-labeled RNA probes specific for the small EBV-encoded nuclear RNAs, EBER1 and EBER2, in some instances preceded by immunohistology for CD20, CD30, CD45RO, and CD68 antigens, the T-cell receptor beta-chain, and latent membrane antigen (LMP) of EBV. Twenty-three of 46 HD cases displayed EBER transcripts in all Hodgkin and Reed-Sternberg (H-RS) cells, and 18 of these cases showed LMP expression exclusively in neoplastic cells. EBER+ small reactive cells were present in 39 cases in low numbers, and in three cases in abundance. Thus, presence of H-RS cells with or without LMP expression was not accompanied by an unrestricted proliferation of reactive EBER+/LMP- lymphoid cells in the majority of HD patients. Simultaneous in situ hybridization with [35S]-labeled immunoglobulin light chain (IgLC) gene probes and nonisotopically labeled EBER probe showed a phenotype of mature B lymphocytes and a polyclonal composition for a large proportion of the EBER+ small cells. However, in contrast to noninfected cells, CD20 expression was not detectable in many of these cells, which may indicate downregulation of certain differentiation antigens in latently EBV-infected small lymphoid cells in vivo.