In situ hybridization (ISH) has emerged over the past decade as an extraordinarily sensitive technique for the detection of gene expression at the cellular level. Advances in probe preparation and labeling methods have facilitated the transfer of this technology from the research laboratory to the clinical arena. In contrast to immunohistochemistry, which is dependent on the protein content of cells, ISH analyses permit the identification of cells on the basis of their contents of specific messenger RNAs (mRNAs) encoding the products of interest. These methods provide a critical approach for the analysis of heterogeneity in tumors that typically contain cells at different phases of neoplastic progression and at multiple levels of differentiation and functional activity. In situ hybridization methods have been of particular value for studies of mRNAs encoding oncogenes, hormones, secretory proteins, cytokines, and a wide variety of other cellular products. Advances in ISH technology, including polymerase chain reaction (PCR) based methods, offer particular promise for examining genes with low levels of expression at the cellular level.