This paper demonstrates that (a) differences in the methylation levels of interphase nuclei can be measured on a cell-by-cell basis, (b) the binding sites of beta-satellite DNA and 5-methylcytosine (5MeC)-rich regions can be localised in interphase nuclei and metaphase chromosomes by sequential in situ hybridization and indirect immunolabelling, and (c) quantitative differences in the relative extensions of beta-satellite DNA and anti-5MeC antibody binding areas can also be measured. This goal was achieved by indirect immunolabelling by anti-5MeC antibodies (Reynaud et al.: Cancer Lett. 61:255-262, 1991) of control and 5-azacytidine-treated human cell cultures. A quantitative analysis of the number, total, and mean areas of labelled heterochromatic regions and the optical densities of euchromatin and heterochromatin was performed for the cells on microscope slides. Dedicated software was used to select and measure the areas of cytological interest. In additional experiments, DAPI-stained slides from control cultures were sequentially treated by in situ hybridization with beta-satellite DNA probe and indirect immunofluorescent labelling with anti-5MeC antibodies. Fluorescent signals of probe and antibodies were pseudocoloured and merged on digital images. The relative locations of probe- and antibody-positive areas were analysed on metaphases and nuclei, and their extensions were quantified in interphase nuclei. Our results show that (a) our analysis can successfully detect different levels of DNA methylation within individual nuclei, (b) in metaphase chromosomes the antibody binding sites are mostly coincident with the hybridisation sites, and (c) in interphase nuclei a quite different picture is consistently observed.