C-1027 belongs to the family of chromoprotein antitumor antibiotics, which contain a carrier apoprotein and a highly unstable enediyne chromophore. The enediyne spontaneously aromatizes to generate p-benzyne biradical, and subsequently abstracts hydrogens from the DNA sugar backbone, resulting in cleavage of the double strand. Using spin-trapping methods, we obtained direct proof of radical intermediates during an DNA cleavage, and found intriguing difference in behavior between the trapping agents 2-methyl-2-nitrosopropane (MNP) and 5,5-dimethyl-1-pyrroline N-oxide (DMPO): MNP added to the sugar radicals of the DNA, whereas DMPO directly trapped a phenyl radical or p-benzyne biradical derived from the C-1027 chromophore.