Copper [Cu(II)] has been shown to enhance DNA damage in several biological systems. Binding of copper to DNA may be a key step in producing these lesions. The results of this study indicate that the DNA double helix contains at least two kinds of binding sites for copper. One site is present once every four nucleotides, has high affinity, and shows a cooperative effect. The other is an intercalating site for copper that is present in every base pair. This site is saturable, has a dissociation constant (Kd) for Cu(II) of 41 microM. In single-stranded DNA, we found an average copper binding site every three nucleotides with lower affinity than in dsDNA. The binding of copper to DNA shows an unexpected high specificity when studied in the presence of other metallic ions. The relative efficacy of several divalent cations to antagonize Cu(II) binding was: Ni = Cd = Mg much much greater than Zn = Hg greater than Ca greater than Pb much much greater than Mn, while Cr(VI) enhanced Cu(II) binding to DNA. We hope this study will broaden the understanding of copper-DNA interactions, particularly as they relate to treatment modalities for diseases associated with disruption of copper homeostasis and potential development of copper antitumor agents.