The authors describe a highly sensitive and practical in situ hybridization method using an oligonucleotide probe for EBER1 RNA for the detection of Epstein-Barr virus (EBV) in formalin-fixed, paraffin-embedded tissue sections. Paraffin-embedded tissues from 793 cases of normal and neoplastic tissues were studied. Nuclear staining for EBV RNA was uniformly present in all or virtually all neoplastic cells in a variety of known EBV-positive tumors. We also demonstrate rare EBV-infected cells in normal lymphoid tissues. RNAase predigestion, competitive inhibition, and control probe studies confirmed the specificity of the staining. In addition, cross-reactivity of EBV RNA staining with other viruses was not present. Additionally, the distribution of EBV in a wide variety of other normal and neoplastic tissues is reported.