The Escherichia coli Orf17 (NtpA, NudB) protein, a MutT-type enzyme, hydrolyzes oxidized deoxyribonucleotides, including 8-hydroxy-2'-deoxyadenosine 5'-triphosphate and 8-hydroxy-2'-deoxyguanosine 5'-triphosphate, in vitro. To examine its in vivo role(s) in bacteria, plasmid DNAs containing the orf17 gene in the sense and antisense orientations were introduced. When the Orf17 protein was overexpressed in mutT cells, the rpoB mutant frequency was decreased. On the other hand, similar effects were not observed when Orf17 was overexpressed in wild type and orf135 cells. Expression of the antisense RNA of the orf17 gene did not produce an obvious phenotype, such as increased mutant frequency and resistance to ionizing radiation. These results suggest that the role of the Orf17 protein is to back up the MutT function, and to assist in the elimination of 8-hydroxy-2'-deoxyguanosine nucleotides.