Tetrahydrobiopterin (BH₄) prevented an increase in the formation of 8-hydroxydeoxyguanosine (8-OHdG) in calf thymus DNA by the peroxynitrite (ONOO^-) or Fe²⁺ + hydrogen peroxide (H₂O₂) system. But, the addition of BH₄ to the Cu²⁺ + ascorbic acid (AsA) system caused a very large increase in base damage. Without the addition of AsA (in the presence of Cu²⁺ alone), BH₄ could also induce 8-OHdG formation. Experiments utilizing the Cu⁺ chelator bathocuproine, catalase and superoxide dismutase revealed that Cu⁺ and H₂O₂ intermediates relate to Cu²⁺ + BH₄-induced 8-OHdG formation. Electron spin resonance experiments also showed that the hydroxyl radical is not a major intermediate. These results suggest that although BH₄ is known to prevent oxidative damage in proteins and lipids, it has the potential to cause oxidative DNA damage depending on the existence of copper ions.