When supercoiled plasmid DNA was incubated with 2,2'-azobis (2-amidinopropane)hydrochloride (AAPH) at pH 7.4 in the presence and absence of oxygen, the DNA single strands were effectively cleaved. The breaking in the presence of oxygen was not inhibited by superoxide dismutase and catalase, but inhibited by mannitol, ethanol, butyl hydroxyanisole, thiol compounds, tertiary amines and spin trapping agents N-tert-butyl-alpha-phenylnitrone (PBN) and 5,5-dimethyl-1-pyrroline N-oxide (DMPO). The breaking in the absence of oxygen was inhibited by ethanol, a tertiary amine and PBN. By electron spin resonance spin-trapping with PBN, the carbon-centered radical was detected both in the presence and the absence of oxygen. Hydroxyl radical was detected by use of DMPO only in the presence of oxygen. The DNA breaking activity of AAPH was found to be due primarily to the aliphatic carbon-centered radical. While the reactivity of carbon-centered radicals have received little attention, the aliphatic carbon-centered radical generated from AAPH was found to be highly reactive to break the DNA strands.