Thymine glycol, a DNA lesion produced by ionizing radiation, has been introduced site specifically at high frequency into a synthetic oligonucleotide by chemical oxidation of the single thymine residue within the sequence. The lesion-containing template was then annealed to a complementary synthetic primer and used to study the effects of cis-thymine glycol lesions on DNA polymerase function in vitro. Synthesis by polymerase I (Klenow fragment), T4 DNA polymerase, and polymerase alpha 2 was arrested quantitatively at the site of the lesion. AMV reverse transcriptase was less inhibited and was able to synthesize past a significant fraction of the lesions. Changing the template base immediately 5' to thymine glycol from A to C did not significantly alter the pattern of synthesis arrest for any of the polymerases. The correct nucleotide, dAMP, was inserted opposite the lesion more than 90% of the time by all four polymerases, suggesting that thymine glycol forms a reasonably stable base pair with adenine. However, the 3'-5' exonuclease activity of polymerase I removed a 3'-terminal dAMP residue more rapidly from an A . thymine glycol base pair than from an A.T base pair. These results suggest that increased nucleotide turnover at the site of the lesion contributes to the inhibitory effects of thymine glycol lesions on DNA synthesis in vitro, at least for polymerases such as polymerase I that have intrinsic or associated editing exonuclease functions.