The chemical nuclease 1,10-phenanthroline-copper cleaves DNA by oxidative attack on the deoxyribose moiety yielding 3'- and 5'-phosphomonoesters, free purine and pyrimidine, and 5-methylenefuranone as stable products. Kinetic isotope effects associated with deuterium substitution have been measured in an attempt to analyze the chemical mechanism of the scission reaction. A kinetic isotope effect of 2.7 is observed with completely perdeuterated DNA, which is substituted in the oxidatively sensitive deoxyribose moiety as well as in the bases. Surprisingly, no isotope effect is found upon cleavage of DNA deuterated in the thymidines at either C-1', C-2',2", or C-4', all positions from which hydrogen is lost during the course of the reaction, by either the 2:1 or the 1:1 1,10-phenanthroline-cuprous complexes. These results suggest that perdeuteration of DNA alters the ligand binding and/or conformational flexibility of the nucleic acid.