A method for identification of a protein-binding region in a double-stranded DNA fragment. One 5'-end of the DNA fragment is labelled with ³²P and the DNA-fragment-protein complex is treated with a 3'-exonuclease to digest from the 3'-end until it meets the region protected by the DNA-binding protein. The labelled strand is characterised subsequently by its size to indicate the distance of the site of the binding protein from the 5'-end of the fragment. The technique is suitable for localization of a binding site to a specific region of a large DNA fragment, whereas footprinting gives the exact sequence of the binding site of a smaller fragment.