We synthesized a dinucleoside monophosphate of the (15)N-labeled (6-4) photoproduct, which is one of the major UV-induced lesions in DNA, to investigate the (6-4) photolyase repair mechanism, and characterized its protonation state by measuring (15)N NMR spectra as a function of pH. We expected that chemical-shift changes of the pyrimidone (15)N3, due to protonation, would be observed at pH 3, as observed for the (15)N-labeled 5-methylpyrimidin-2-one nucleoside. Interestingly, however, the changes were observed only in alkaline solutions. In UV absorption spectroscopy and HPLC analyses under acidic conditions, a change in the maximum absorption wavelength, due to the protonation-induced hydrolysis, was observed at and below pH 1, but not at pH 2, whereas the protonation of 5-methylpyrimidin-2-one occurred at pH values between 2 and 3. These results indicated that the pK(a) value for this N3 is remarkably lower than that of a normal pyrimidone ring, and strongly suggest that an intramolecular hydrogen bond is formed between the N3 of the 3' base and the 5-OH of the 5' base under physiological conditions. The results of this study have implications not only for the recognition and reaction mechanisms of (6-4) photolyase, but also for the chemical nature of the (6-4) photoproduct.