DNA glycosylases function to remove endogenous and exogenous base damage and thus contribute to the maintenance of genomic integrity. This function gains clinical relevance when base mispairs introduced by chemotherapy or radiosensitizing drugs become their substrate. This report describes the action of DNA glycosylases on the mispairs generated by iododeoxyuridine (IUdR)-a radiosensitizer. A non-radioactive fluorescent dye-based in vitro glycosylase assay was employed to quantitatively measure the enzymatic activities of functionally related DNA glycosylases on IUdR generated mispairs including G:IU and A:IU. Thymine DNA glycosylase (TDG) and methyl binding domain protein 4 (MBD4/MED1) are found to act on G:IU (but not A:IU) mispairs and are functionally complementary to each other. However, uracil DNA glycosylase (UDG) does not show any activity on these mispairs. The methyl binding domain of MBD4/MED1 was found to specifically inhibit the activity of MBD4/MED1 as well as the glycosylase domain, when the G:IU mispairs were located in a methylated CpG context. However, inhibition of TDG activity on methylated G:IU mispairs by the methyl binding domain was not observed.