An immunodetection technique has been developed to map with high resolution the methylated sites of human chromosomes. We have used this method to define the methylated areas of chromosomes from normal donors and from leukemia cell lines. The chromosomes were exposed for a short time to UV light to induce mild denaturation. The methylated sites were detected in situ by using monoclonal antibodies against 5-methylcytosine (prepared in mouse), and fluorescein-conjugated antimouse immunoglobulins. The chromosomes from normal cells exhibited a fluorescent pattern with RCT banding, although some differences from previously reported patterns could be detected. With this method we have been able to show the presence of two types of R-bands: High fluorescence R-band (HFR) and low fluorescence R-band (LFR). Chromosomes from leukemia cell lines exhibited low global staining with disrupted RCT banding of the chromosomes. The decreased level of the methylation status of the chromosomes from leukemia cells was confirmed by detection of 5-methylcytosines on total immobilized DNA. Thus, we have shown that this method can be used to determine the methylated status of chromosomes and, in turn, to map not only the structural (banding) but also the functional (methylation status) properties of the different chromosome domains in normal and pathologic human cells.