Chemiluminescence is the light emission produced by a chemical reaction in which chemically excited molecules decay to the ground state. The phenomenon is utilized in various analytical techniques in which small amounts of analytes or enzymes can be detected and quantified by measurement of the light emitted by bio- or chemiluminescent reactions. Recently chemiluminescence has been proposed as a valid alternative to radioactive or colorimetric methods in in situ hybridization assays, in which target nucleic acids are localized by labeled probes inside individual cells with the preservation of cell morphology. Chemiluminescence in situ hybridization is performed using probes that are detected using enzymes with their appropriate chemiluminescent substrates. The luminescent signal from the hybrid formation is detected, analysed and measured with a high performance low light level imaging apparatus connected to an optical microscope and to a personal computer for quantitative image analysis. Generally, the instrumental system to detect positive signals after in situ hybridization operates in three steps: firstly tissue structures and cells are recorded in transmitted light then the luminescent signal is measured with an optimized photon accumulation; and then, after a computer elaboration of the luminescent signal with pseudocolors corresponding to the light intensity, an overlay of the two images on the screen provided by the transmitted light and by the luminescent signal allows the spatial distribution of the labeled probe to be localized and evaluated. The main advantages of chemiluminescence in situ hybridization are mainly the sensitivity, the quantification of the data, the objectivity of the evaluation and the digital imaging of the results. The chemiluminescence in situ hybridization assay, which can be applied to cell smears, archival frozen and paraffin embedded tissue samples, can be a useful tool for a sensitive and specific diagnosis of viral infections and for the detection and study of specific genic sequences inside the cells. The use of the chemiluminescent in situ hybridization assay is also promising for an estimation and quantification of nucleic acids present in tissue samples or cellular smears and for imaging gene expression in cells.