The compound [Cu-2(II)(D-1)(H2O)(2)](ClO4)(4) (D-1 = dinucleating ligand with two tris(2-pyridylmethyl)-amine units covalently linked in their 5-pyridyl positions by a -CH2CH2-bridge) selectively promotes cleavage of DNA on oligonucleotide strands that extend from the 3' side of frayed duplex structures at a site two residues displaced from the junction. The minimal requirements for reaction include a guanine in the n (i.e. first unpaired) position of the 3' overhang adjacent to the cleavage site and an adenine in the n position on the 5' overhang. Recognition and strand scission are independent of the nucleobase at the cleavage site. The necessary presence of both a reductant and dioxygen indicates that the intermediate responsible for cleavage is produced by the activation of dioxygen by a copper(l) form of the dinuclear complex. The lack of sensitivity to radical quenching agents and the high level of site selectivity in scission suggest a mechanism that does not involve a diffusible radical species. The multiple metal center exhibits a synergy to promote efficient cleavage as compared to the action of a mononuclear analogue [Cu-II(TMPA)-(H2O)](ClO4)(2) (TMPA = tris(2-pyridylmethyl)amine) and [Cu(OP)(2)](2+) (OP = 1, 10-phenanthroline) at equivalent copper ion concentrations. The dinuclear complex, [Cu-2(II)(D-1)(H2O)(2)](ClO4)(4), is even capable of mediating efficient specific strand scission at concentrations where [Cu(OP)(2)](2+) does not detectably modify DNA. The unique coordination and reactivity properties of [Cu-2(II)(D-1)(H2O)(2)](ClO4)(4) are critical for its efficiency and site selectivity since an analogue, [Cu-2(II)(DO)(Cl-2)](ClO4)(2), where DO is a dinucleating ligand very similar to D-1, but with a -CH2OCH2- bridge, exhibits only nonselective cleavage of DNA. The differences in the reactivity of these two complexes with DNA and their previously established interaction with dioxygen suggest that specific strand scission is a function of the orientation of a reactive intermediate.