The mutY homolog gene (mutY_Dr) from Deinococcus radiodurans encodes a 39.4-kDa protein consisting of 363 amino acids that displays 35% identity to the Escherichia coli MutY (MutY_Ec) protein. Expressed MutY_Dr is able to complement E. coli mutY mutants but not mutM mutants to reduce the mutation frequency. The glycosylase and binding activities of MutY_Dr with an A/G-containing substrate are more sensitive to high salt and EDTA concentrations than the activities with an A/7,8-dihydro-8-oxoguanine (GO)-containing substrate are. Like the MutY_Ec protein, purified recombinant MutY_Dr expressed in E. coli has adenine glycosylase activity with A/G, A/C, and A/GO mismatches and weak guanine glycosylase activity with a G/GO mismatch. However, MutY_Dr exhibits limited apurinic/apyrimidinic lyase activity and can form only weak covalent protein-DNA complexes in the presence of sodium borohydride. This may be due to an arginine residue that is present in MutY_Dr at the position corresponding to the position of MutY_Ec Lys142, which forms the Schiff base with DNA. The kinetic parameters of MutY_Dr are similar to those of MutY_Ec. Although MutY_Dr has similar substrate specificity and a binding preference for an A/GO mismatch over an A/G mismatch, as MutY_Ec does, the binding affinities for both mismatches are slightly lower for MutY_Dr than for MutY_Ec. Thus, MutY_Dr can protect the cell from GO mutational effects caused by ionizing radiation and oxidative stress.