We studied the yield and distribution of DNA strand breaks produced by decay of 125I introduced into triplex-forming and duplex-forming oligodeoxyribonucleotide (ODNs) through linkers of various lengths. ODNs were prepared with 125I attached at the 5'-end with a long linker or to an internal nucleotide position with a short linker. The 125I-ODNs were hybridized to either a single-stranded target to form duplexes or to a double-stranded target to form triplexes. After decay accumulation, the duplex and triplex samples were assayed for strand breaks in a sequencing gel. The yield of strand breaks per decay was 0.34 for duplex with the 5'-modified ODN and 0.66 for duplex with internally modified ODN. The triplex samples with internal 125I have different yields of DNA breaks in the pyrimidine and purine strands, 0.16 and 0.37, respectively. The yield of DNA breaks in the pyrimidine strand of the triplex with the 5'-modified ODN is 0.46. The majority of breaks are located within 5 nucleotides from the decay site. The yield of strand cleavage per decay of 125I was nearly two-fold lower with the described linkers in comparison with the results obtained when 125I is directly attached to the C-5 position of cytosine. Nevertheless, the rapid iodination procedure reported here combined with the possibility of multiple incorporations of 125I on the linkers makes such 125I-ODNs promising agents for sequence-specific cleavage of DNA.