The reactivity of two [peptide-Cu] complexes ([GGH-Cu](-) and [KGHK-Cu](+)) toward DNA cleavage has been quantitatively investigated. Neither complex promoted hydrolytic cleavage, but efficient oxidative cleavage was observed in the presence of a mild reducing agent (ascorbate) and dioxygen. Studies with scavengers of ROS confirmed hydrogen peroxide to be an obligatory diffusible intermediate. While oxidative cleavage of DNA was observed for CU2+(aq) under the conditions used, the kinetics of cleavage and reaction products/pathway were distinct from those displayed by [peptide-Cu] complexes. DNA cleavage chemistry is mediated by the H2O-dependent pathway following C-4'H abstraction from the minor groove. Such a cleavage path also provides a ready explanation for the linearization reaction promoted by [KGHK-Cu]+. Kinetic activities and reaction pathways are compared to published results on other chemical nucleases. Both [peptide-Cu] complexes were found to display second-order kinetics, with rate constants k(2) similar to 39 and 93 M-1 s(-1) for [GGH-Cu](-) and [KGHK-Cu](+), respectively. Neither complex displayed enzymelike saturation behavior, consistent with the relatively low binding affinity and residence time expected for association with dsDNA, and the absence of a prereaction complex. However, the intrinsic activity of each is superior to other catalyst systems, as determined from relative k(2) or k(cat)/K-m values. Linearization of DNA was observed for [KGHK-Cu](+) relative to [GGH-Cu](-), consistent with the increased positive charge and longer residency time on dsDNA.