The (6-4) photoproduct, which is one of major UV-induced lesions formed between adjacent pyrimidine bases in DNA, was characterized by using (15)N NMR, fluorescence emission, and UV/VIS absorption at various pH values. From these experiments, we identified a novel intramolecular hydrogen bond between the hydroxyl group at the C5 position of the 5' component and the N3 of the 3' pyrimidone ring within the (6-4) photoproduct.