MutT protein of Escherichia coli prevents the occurrence of A:T --> C:G transversion by hydrolyzing an oxidized form of dGTP, 8-oxo-7, 8-dihydro-2'-deoxyguanosine 5'-triphosphate (8-oxo-dGTP), which is produced by active oxygen species. In a search for mutT-related genes, we found that the ribA gene, encoding GTP cyclohydrolase II, is able to reduce the increased level of mutation frequency of the mutT strain to almost the normal level, provided that the gene product is overproduced. Purified preparations of Escherichia coli GTP cyclohydrolase II protein as well as the histidine hexamer-tagged recombinant GTP cyclohydrolase II protein efficiently hydrolyze 8-oxo-dGTP and 8-oxo-GTP, producing 8-oxo-dGMP and 8-oxo-GMP, respectively. dGTP was not hydrolyzed by these preparations. GTP cyclohydrolase II catalyzes conversion of GTP to 2, 5-diamino-6-hydroxy-4-(5-phosphoribosylamino)-pyrimidine, which constitutes the first step for riboflavin synthesis. The Km values for the three types of guanine nucleotides, GTP, 8-oxo-GTP, and 8-oxo-dGTP, were almost the same. In the mutT- background, ribA- cells showed higher spontaneous mutation frequencies as compared with that of ribA+ cells. Thus, GTP cyclohydrolase II, the ribA gene product, has a potential to protect genetic material from the untoward effects of endogenous oxygen radicals.