5,6-Dihydroxy-5,6-dihydrothymine (thymine glycol) is formed in DNA by reaction with oxidizing agents and as a result of ionizing and near-ultraviolet radiation. We describe a rapid purification of cis-5,6-dihydroxy-5,6-dihydrothymine and cis-5,6-dihydroxy-5,6-dihydrothymidine (cis-thymidine glycol) and their use as markers in identifying the thymine glycol moiety in oxidized DNA. Both glycols were prepared by oxidation of [14C]thymine and -thymidine with KMnO4 followed by purification on Sephadex LH-20 (LH-20). [3H]DNA was oxidized with KMnO4 and the thymidine glycol in DNA identified by enzymatic digestion of the DNA followed by cochromatography of the digest with marker [14C]thymidine glycol on LH-20. The cis conformation of the glycol was confirmed by the change in the elution pattern when borate rather than water was used as eluent. Alkaline hydrolysis of a mixture of [14C]thymine glycol and oxidized [3H]DNA followed by trichloroacetic acid precipitation and LH-20 chromatographic analysis of the neutralized supernatant yielded a complex pattern of radioactive degradation products with coincidence of one 14C marker- and one [3H]-DNA-derived peak. All applied radioactivity was recovered. This methodology should be useful in determining thymine glycol content of irradiated DNA and in elucidating the mechanism by which these altered residues are removed from cellular DNA by repair enzymes.