The C-8 position of deoxyguanosine (dGuo) was hydroxylated by ascorbic acid in the presence of oxygen (O2) in 0.1 M phosphate buffer (pH 6.8) at 37°C. Addition of hydrogen peroxide (H ₂ O ₂ ) remarkably enhanced this hydro-xylation. The Udenfriend system [ ascorbic acid, Fe ^II , ethylenediaminetetra-acetic acid (EDTA) and 02] was also effective for hydroxylation of dGuo in high yield. Guanine residues in DNA were also hydroxylated by ascorbic acid. Other reducing agents, such as hydroxylamine, hydrazine, dihydroxymaleic acid, sodium bisulfite and acetol, were also effective for the hydroxylation reaction, as were metal-EDTA complexes (Fe ^II -, Sn ^II Ti ^III -, Cu ^I -EDTA) An OH radical seemed to be involved in this hydroxylation reaction in most of the above hydroxylating systems, but another reaction mechanism may also be involved, particularly when dGuo is hydroxylated by ascorbic acid alone or ascorbic acid plus H ₂ O ₂ The possible biological significance of the hydroxylation of guanine residues in DNA in relation to mutagenesis and carcinogenesis is discussed.