A sensitive and specific radioimmunoassay for 5,6-dihydroxy-5,6-dihydrothymine (thymine glycol) was developed. As little as 4 femtomoles of thymine glycol can be detected in ${\rm O}_{{\rm s}}{\rm O}_{4}\text{-treated}$ DNA. Unmodified DNA cross-reacts only at the highest concentrations. The assay is dependent on both the absolute amount of thymine glycol and the fraction of thymine altered in the polymer. This is caused by the high specificity of the antibody for multiple thymine glycol sites, as confirmed using ${\rm O}_{{\rm s}}{\rm O}_{4}\text{-modified}$ poly (dA-dT) and a series of oligothymidylic acids. A method of correcting for this dependence is presented. Using this RIA thymine glycol is detected in γ-irradiated DNA at a G value of 0.018; the reasons for this low yield are discussed. The assay requires only mild chemical treatment and many samples can be quickly and easily handled.