Two single-copy DNA segments of 6 kilobases (kb) and 2.3 kb were labeled with biotin-labeled dUTP (Bio11-dUTP) and hybridized to human chromosomes. These probes were detected by immunofluorescence and directly mapped on chromosomes by using classical fluorescence microscopy and a microchannel-plate-intensified video camera. By a subsequent R-banding, the 6-kb and 2.3-kb fragments were precisely localized to the 18p11.3 band and to the 22q11.2 band, respectively, in agreement with previous results obtained with radioactive probes. The adaptation of fluorescence intensification and digital image processing (frame integration to enhance signal-to-noise ratio and linear contrast stretching) to microscopy makes it possible to detect very weak fluorescent spots on chromosomes. This system allows a high spatial resolution (less than 0.6 micron), even at very low fluorescence levels. The efficiency and the specificity of the hybridization and detection methodology give a direct and precise localization of the short single-copy sequences on human chromosomes.