In tumors, DNA is often globally hypomethylated compared to DNA extracted from normal tissues. This observation is usually made after extraction and exhaustive digestion of DNA followed by analysis of nucleosides by chromatography or digestion with restriction enzymes, gel analysis, and hybridization. This approach provides an average value which does not give information on the various cell subpopulations included in heterogeneous samples. Therefore an immunochemical technique was set up with the aim of demonstrating, in a population of mixed cells, the possibility of detecting the presence of individual nuclei containing hypomethylated DNA, on a cell-by-cell basis. Monoclonal antibodies to 5-methylcytidine were used to label cells grown in vitro. Under appropriate fixation and permeabilization conditions, interphase nuclei were labeled. Quantitative differences in the labeling were detected between Epstein-Barr virus-transformed cells and normal peripheral blood monocytes by flow cytometry analysis. Similar differences were observed by fluorescence microscopy. Both results were confirmed by Southern transfer and hybridization of DNA fragments generated by restriction enzyme digestion. This observation, which is in accordance with the occurrence of global DNA hypomethylation in tumors as established by chromatography, opens the field for the analysis of fresh tumor samples by flow cytometry and microscopy.