Epstein-Barr virus (EBV) nucleotide sequences can be detected in routinely processed tissues by in situ hybridization (ISH) with either radiolabeled or nonisotopic EBV probes. In this paper, we describe our nonisotopic ISH protocol for EBV detection with use of a triple-biotinylated oligonucleotide NotI/PstI EBV probe. We compared this technique with our radioisotopic ISH technique that uses a 35S-labeled EBV probe and found that the nonisotopic technique was as sensitive as the radioisotopic technique for detecting EBV-infected cells in tissues from patients with posttransplantation lymphoproliferative disorders. Furthermore, use of the biotinylated probe has several advantages over the 35S-labeled probe, including commercial availability of labeled probe, increased probe stability, decreased technical and development time, and ease of interpretation. We conclude that the nonisotopic ISH procedure is practical for use in diagnostic surgical pathology.