alpha-Deoxynucleotides are potentially deleterious lesions when produced in DNA. They are presumably formed in part via misrepair of the respective C1'-nucleotide radicals by thiols. However, the selectivity and extent to which these lesions are formed via this pathway has not been ascertained. Using the ability to independently generate deoxyuridin-1'-yl (4) at a defined site in a biopolymer, we have determined that thiol trapping in duplex DNA occurs with high stereoselectivity from the ct-face, resulting in restoration of the naturally occurring beta-deoxynucleotide. The observed stereoselectivity of thiol trapping in duplex DNA suggests that 4 is intrahelical. The rate constant for hydrogen atom donation to 4 is reduced 2-3-fold in double-stranded DNA compared to single-stranded DNA. This decrease is attributed to the relative inaccessibility of the C1'-position in duplex DNA. The combination of these two properties of 4 indicates that, at O-2 concentrations present in aerated water, alpha-deoxynucleotide formation should constitute a minor component of the reactivity of C1'-radicals. Accordingly, the chemical biology of other lesions derived from formal damage at C1'-position could be significant.