The DNA base sequence specificity of the 64M-1 monoclonal antibody, which recognizes ultraviolet (UV)-induced (6-4)photoproducts, was characterized. The 64M-1 antibody strongly bound to UV-poly(dU) as well as to UV-poly(dT), and weakly to UV-poly(dC), UV-poly(me5dC) and UV-poly(rU). A competitive inhibition assay using UV-oligo(dT)8, UV-oligo(dTdC)4, UV-oligo(dC)8, UV-PvuI linker (GCGATCGC) and UV-PvuII linker (GCAGCTGC) indicated that the main (6-4)photoproducts detected by the 64M-1 antibody in UV-irradiated DNA are TT(6-4)photoproducts and TC(6-4)photoproducts. Comparison between dTpdT(6-4)photoproduct and dTpdC(6-4)photoproduct showed that the affinity of the 64M-1 antibody for dTpdT(6-4)photoproduct was about 5 times higher than that for dTpdC(6-4)photoproduct. The antibody also binds to isolated TT(6-4)photoproducts.