Oxidation of the thymine methyl group can generate 5-formyluracil (FoU), which is known to be both mutagenic and chemically unstable in DNA. Synthetic oligonucleotides containing FoU at defined sites have been prepared to investigate potential mechanisms by which FoU might perturb DNA function. The half-life of the glycosidic bond of an FoU residue in single-stranded DNA under physiological conditions of temperature and pH is estimated to be approximately 148 days, orders of magnitude shorter than the parent pyrimidine, thymine. This reduced stability of FoU residues in DNA is attributed to the inductive properties of the 5-formyl substituent. Oxidative modification of the thymine methyl group could also inhibit association with sequence-specific DNA-binding proteins. Alternatively, the 5-formyl substituent of FoU could cross-link nonspecifically with protein amino groups. Transcription factor AP-1 is known to make specific contacts with thymine methyl groups of DNA in its recognition sequence. Substitution of T by FoU is shown to inhibit AP-1 (c-Jun homodimer) binding with a DeltaDeltaG of approximately 0.6 kcal/mol. No evidence of cross-link formation is observed with either AP-1 or polylysine. Molecular modeling studies on the FoU-containing oligonucleotide sequence corresponding to the duplex used in the experimental studies demonstrate that the 5-formyl substituent of an FoU residue paired with adenine lies in the plane of the pyrimidine base and is well protected from solvent on one face and only partially accessible on the other. The results of this study suggest that although FoU residues in DNA are considerably more labile than thymine, they are likely to be present long enough to miscode as well as interfere with DNA-protein interactions.