The enzymatic repair of O4-methyldeoxythymidine (O4-MedT) by mammalian liver extracts was investigated in vitro using double-stranded poly[d(A-T).d(A-T)] alkylated with N-[methyl-3H]-N-nitrosourea as a substrate. The removal of O4-MedT by monkey liver extracts was proportional to the protein concentration of the reaction mixture up to ∼4 mg/ml, while kinetic analysis revealed the repair reaction reached a plateau by 2 h at 37°C. The removal of O4-MedT could not be accounted for by contaminating nuclease activity, for >90% of the 3-methyldeoxythymidine (3-MedT) was always recovered, whereas O4-MedT declined to as little as 25% of control values. A DNA-glycoslyase is not involved in the repair of O4-MedT, for O4-methylthymine, the free base, was not detected in supernatants from monkey liver reaction mixtures following incubation with substrate and precipitation of macromolecules with ethanol, even though analysis of nucleic acid digests of these assays revealed O4-MedT was lost from the substrate. The order of O4-MedT repair activity in human, monkey, partially hepatectomized rat and methylguanine DNA-methyltransferase activity, with human and monkey most active, partially hepatectomized rat intermediate in activity and control rat least active. Our findings suggest the same, or a similarly regulated mammalian liver DNA-methyltransferase repairs O6-methylguanine and O4-MedT residues in DNA, yet the affinity of the enzyme(s) differs for these promutagenic adducts, as we estimate 30–50 O6-methylguanines would be repaired for every one O4-MedT repaired.