The mutational specificity and genetic requirements for mutagenesis by 5,6-dihydroxy-5,6-dihydrothymine (thymine glycol), one of the principal DNA lesions induced by oxidation and ionizing radiation, has been investigated in Escherichia coli. Thymine glycol was positioned at a unique site in the single-stranded genome of a bacteriophage M13mp19 derivative. Replication of the genome in E. coli yielded targeted mutations at a frequency of 0.3%; the mutations were exclusively T----C. Mutagenesis was independent of SOS and nth (nth encodes endonuclease III, a thymine glycol repair enzyme). The adduct was not detectably mutagenic in duplex DNA. A chemical rationalization for the mutation observed for thymine glycol was developed by applying molecular modeling and molecular mechanical calculations to the same DNA sequence studied in vivo. Modeling suggested that the 5R,6S isomer of cis-thymine glycol, when not base paired, was displaced laterally by approximately 0.5 A toward the major groove in comparison to the position that thymine would otherwise occupy. This perturbation of DNA structure should increase the likelihood of a guanine.thymine glycol wobble base pair during replication, which would explain the mutational specificity of the base observed in the genetic experiments.