In situ hybridization (ISH) is a technique by which specific nucleotide sequences are identified in cells or tissue sections. These may be endogenous, bacterial or viral, DNA or RNA. On the basis of research applications, the technique is now being translated into diagnostic practice, mainly in the areas of gene expression, infection and interphase cytogenetics. Diagnostic applications are most often based on short nucleotide sequences (oligomers) labelled with non-isotopic reporter molecules, and sites of binding may be localized by histochemical or immunohistochemical methods. The technique can be applied to routinely fixed and processed tissues; with some targets, it is even possible to obtain hybridization in autopsy material. ISH has been used to detect messenger RNA (mRNA) as a marker of gene expression, where levels of protein storage are low; for example, to confirm an endocrine tumour as the source of excess hormone production. Its application in infectious diseases has to date been mainly in viral infections, such as the typing of human papillomavirus (HPV) or the detection of Epstein-Barr virus by the presence of small nuclear RNAs (EBERs). The expression of mRNAs for histone proteins has been used to detect cells in S phase, and related methods may be applied to detect apoptotic cells. Using probes to chromosome-specific sequences, it is possible to detect aneuploidy, and to document changes in specific chromosomes, which may have prognostic significance in some tumours, such as B-cell chronic lymphatic leukaemia. Using sequence-specific probes, translocations can be identified, such as the t(11;12) of Ewing's sarcoma. This review presents an outline of the technique of in situ hybridization and discusses areas of current and potential diagnostic application.