Triplex forming oligonucleotides (TFO) labeled with Auger emitters could be ideal vehicles to deliver radioactive-decay energy to specific DNA sequences, causing DNA breaks and, subsequently, inactivation of these sequences. To demonstrate this approach we labeled with 125I (two 125I per molecule on average) a purine-rich 38-mer which forms a stable triplex with a polypurine x polypyrimidine stretch in the human HPRT gene. Decay of 125I in the bound TFO was shown to cause sequence-specific double strand breaks (DSB) in the target HPRT sequence cloned into plasmid DNA. No sequence-specific breaks were observed if 125I-labeled TFO were not bound to the plasmid DNA. After 60 days of decay accumulation (one 125I half-life) approximately a quarter of all plasmid molecules contained sequence-specific DSB, corresponding to 0.3 site-specific DSB per decay. Sequencing gel analysis shows that the DNA breaks are distributed within a few bases of the maxima at those bases opposite to the positions of 125I in the TFO.