Nucleotide Excision Repair (NER) is a very versatile DNA repair pathway that enables cells to repair UV induced DNA lesions as well as structurally diverse types of adducts formed by carcinogens. Versatility and specificity in NER are achieved through the sequential and highly coordinated action of at least 30 proteins that detect the lesion and excise a damage-containing oligonucleotide of 24 to 32 nucleotides in length followed by repair synthesis and ligation to restore the DNA sequence to its original state. Reconstituted in vitro NER reactions are one of the most powerful ways to investigate this DNA repair pathway. In a typical NER assay, plasmids containing a lesion are incubated with a Hela whole cell extract followed by radioactive labeling of the oligonucleotides after the reaction. Although this technique has been intensively used and has allowed the collection of much data about the NER mechanism, it is limited in scope. Our aim was to overcome those limitations by developing a new substrate which would allow the monitoring of the NER reaction by fluorescent techniques. Toward this objective, we synthesized a structural analogue of the efficient NER substrate 8-acetylaminofluorene-2'-deoxyguanosine (AAF) containing a ketone linker allowing its site-specific labeling with fluorophores or biotin via oxime formation. Following incorporation into DNA this new substrate will alleviate the need for radioactive labeling in NER by using fluorescence as the method of detection of the oligonucleotides released by the NER reaction. Also, when coupled to a fluorophore, the lesion contains an internal label of the released oligonucleotides, which permits the detection and quantification of all the different products of the NER reaction, which is not possible with current labeling techniques. Together with the use of GFP-tagged NER proteins, this new substrate should also allow the monitoring of the interaction between NER proteins and substrate by fluorescent imaging techniques.