We have previously demonstrated that the DNA-binding efficiency of Sp1 is greatly decreased in nuclear extracts from 30-month-old rat tissues compared to those from young ones, although its gene appears to be normally expressed. As reactive oxygen intermediates are known to accumulate in aged animals, we investigated the effect of oxidation on the Sp1 DNA-binding activity. Electrophoretic mobility shift assays and DNase I footprintings showed that high concentrations of dithiothreitol, added to the aged tissue extracts, fully restore the Sp1 DNA-binding efficiency. However, in young nuclear extracts hydrogen peroxide treatment strongly decreases the Sp1 DNA-binding activity that is restored by the treatment with high dithiothreitol concentrations. To ascertain whether the oxidative stress is directed toward the Sp1 molecule alone, or whether it acts on unknown Sp1 cofactor(s) necessary for DNA binding, we purified Sp1 from young rat liver and demonstrated that when the purified protein is added to aged nuclear extracts, it efficiently binds to its DNA cis-element. Moreover, purified Sp1 treated with hydrogen peroxide lost its ability to bind its cis-element and the DNA-binding efficiency was fully restored after incubation with dithiothreitol.