The specificity of the nucleotide sequence of DNA strand cleavage sites produced by dihydropyrazines, a new type of DNA strand-cleaving agent, was studied. Biotin-5'-end-labeled PCR-amplified DNA restriction fragments of different defined nucleotide sequences were prepared from plasmid pBR322, and reacted with dihydropyrazines in the presence of Cu2+. After being heated with aqueous piperidine, the DNA products were analyzed on polyacrylamide gels. The most preferentially cleaved sites induced by dihydropyrazines were at purine/pyrimidine-guanine (5' --> 3') sequences. The purine/pyrimidine-adenine, pyrimidine-pyrimidine and purine-pyrimidine (5' --> 3') sequences were more resistant to attack by these dihydropyrazines. The side chains of the dihydropyrazine skeleton greatly affected the DNA strand-cleaving activity, and also, to some extent, the nucleotide sequence-specificity in the DNA strand-cleavage.