Large-scale hybridization-based genome mapping projects, such as the production of sequence-ready physical clone maps, call for robust and cheap DNA labeling techniques that are amenable to automation. We routinely use a highthroughput protocol based on fluorescence detection. DNA probes are labeled via polymerase chain reaction (PCR) amplification with primers that are digoxigenin-modified at their 5' ends. Alternatively, digoxigenin-labeled dUTP is incorporated in a random hexamer priming reaction. Hybridization takes place in small volumes by sandwiching the probe between filters and plastic sheets. A fluorescence signal is produced by the activity of alkaline phosphatase attached to an anti-digoxigenin antibody upon the addition of AttoPhosTM substrate. Signals are directly detected with a charge-coupled device (CCD) camera and scored by an image data analysis system. DNA filters can be reused at least 40 times without loss of data quality. Significant advantages compared to radioactive techniques are the reduced health risk, enabling highly parallel processing; the production of spot signals uniform in size and intensity, which is essential for efficient image analysis; and a cost reduction of about 70%.