We examined the mechanism of DNA damage induced by a mutagenic tyrosine metabolite, homogentisic acid (HGA), using 32P-5'-end-labeled DNA fragments obtained from the human p53 tumor suppressor gene. HGA caused DNA damage in the presence of Cu(II), particularly at thymine and cytosine residues. Catalase and bathocuproine inhibited the DNA damage, suggesting the involvement of H2O2 and Cu(I). The formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine by HGA increased depending on HGA concentration in the presence of Cu(II). It is concluded that H2O2 is generated during Cu(II)-catalyzed HGA autoxidation and reacts with Cu(I) to form the Cu(I)-peroxide complex, capable of causing oxidative DNA damage.