Aims: Epstein–Barr virus (EBV) has been implicated in thepathogenesis of nasopharyngeal carcinoma and a range ofproliferative lymphoid conditions. In situ hybridisation (ISH)looking for virus-encoded RNA (EBER) transcripts isperformed simply using a commercially available probe.We aimed to examine the application of this test in a routinediagnostic setting.Methods: In total, 26 cases in which EBV ISH wasrequested for diagnostic purposes were examined. Welooked at the indication for testing, the result and itsimplication for the final diagnosis.Results: Cases were classified into three categories:possible nasopharyngeal carcinoma; possible EBV-relatedlymphoma; and possible immunodeficiency-associated lymphoproliferativedisorder. Six of nine cases of possiblenasopharyngeal carcinoma were EBV ISH positive (3/3primary and 3/6 secondary), confirming the diagnosis. Threeof 14 possible lymphoma cases were EBV ISH positivewhich, along with appropriate ancillary tests, assisted inmaking the diagnoses of Burkitt’s lymphoma, lymphomatoidgranulomatosis and extranodal NK/T-cell lymphoma ofnasal type. All of three immunodeficiency-associatedcases were EBV ISH positive. Two of these were posttransplantlymphoproliferative disorders, monomorphic type.The third case was classified as HIV-related polymorphiclymphoproliferative disorder.Conclusions: In our experience, EBV ISH is a straightforwardand rapid procedure to perform, giving unequivocalresults. Used in the appropriate clinicopathological setting itcan be a highly useful ancillary diagnostic aid.