To prepare oligonucleotides containing the pyrimidine(6-4)pyrimidone photoproduct between thymine and cytosine, which is formed more efficiently than any counterpart at other dipyrimidine sites, a dimer building block of this photoproduct was synthesized. Irradiation of thymidylyl(3＇-5＇)deoxycytidine, with protecting groups at the phosphate and the 3＇-hydroxyl function, gave a hydrate of cytosine as the main product, and the formation of the (6-4) photoproduct reached a plateau faster than that from the thymidylyl(3＇-5＇)thymidine derivative. After chromatographic purification of the desired (6-4) photoproduct, a phosphoramidite building block was synthesized in three steps. When this compound was used for the usual oligonucleotide assembly on a solid support, it was found that acylation of the amino group of the 5＇-pyrimidine component occurred at the capping step. Oligonucleotides were synthesized successfully by omitting the capping steps after coupling of the (6-4) photoproduct unit. These synthetic oligonucleotides were applied to the characterization of the (6-4) photolyase. By HPLC analyses of the photoreactivation product and its nucleoside composition, it was demonstrated that this (6-4) photoproduct was repaired to its original pyrimidine components by this enzyme.