To evaluate the effect of base pairing of the target pyrimidine on the interstrand photo-cross-linking reaction of DNA via 3-cyanovinylcarbazole nucleoside ((CNV)K), a complementary base of target pyrimidine was substituted with noncanonical purine bases or 1,3-propandiol (S). As the decrease of the hydrogen bonds in the base pairing of target C accelerated the photo-cross-linking reaction markedly (3.6- to 7.7-fold), it can be concluded that the number of hydrogen bonds in the base pairing, i.e., the stability of base pairing, of the target pyrimidine plays a critical role in the interstrand photo-cross-linking reaction. In the case of G to S substitution, the highest photoreactivity toward C was observed, whose photoreaction rate constant (k = 2.0 s(-1)) is comparable to that of (CNV)K toward T paired with A (k = 3.5 s(-1)). This is the most reactive photo-cross-linking reaction toward C in the sequence specific interstrand photo-cross-linking. This might facilitate the design of the photo-cross-linkable oligodeoxyribonucleotides for various target sequences.