The generation of reactive oxygen species by Co(II) from H2O2 in the presence of chelators and related DNA damage was investigated by electron spin resonance (ESR), electrophoretic assays, and high-performance liquid chromatography (HPLC). Incubation of Co(II) with beta-alanyl-3-methyl-L-histidine (anserine) and H2O2 generated .OH radicals. Omission of any one component sharply reduced the amount of .OH radicals generated, indicating that anserine modulated the oxidation potential of Co(II) to enhance its capability to generate .OH radicals from H2O2. Formate only moderately decreased the .OH radical generation, while ethanol had no observable effect, indicating that the generation of .OH radical is site specific. The metal ion chelator 1,10-phenanthroline reduced the .OH radical generation, and deferoxamine suppressed it with the formation of deferoxamine nitroxide radical. Electrophoretic assays using both lambda Hind III linear DNA and PM2 supercoiled DNA showed that .OH radicals generated from a mixture of Co(II), H2O2, and anserine caused DNA strand breaks. A mixture of Co(II), H2O2, and 1,10-phenanthroline also caused DNA strand breaks, which were inhibited by sodium azide, indicating that 1O2 was involved in DNA damage. HPLC measurements showed that .OH radicals and 1O2 generated by Co(II) reactions caused 2'-deoxyguanosine hydroxylation to form 8-hydroxy-2'-deoxyguanosine. ESR spin trapping measurements provided evidence for 1O2 generation by Co(II) from H2O2 in the presence of 1,10-phenanthroline. The results indicate that the oxidation potential of Co(II) can be modulated by chelators to facilitate its generation of reactive oxygen species from H2O2. These species may be involved in Co(II)-induced cellular damage.