Cellular DNA, RNA and their precursor nucleotides are at high risk of being oxidized by reactive oxygen species. An oxidized base, 8-oxo-7,8-dihydro-2'-(deoxy)guanosine, can pair with both adenine and cytosine, and thus would cause both replicational and translational errors. Previously, we have reported that an Arabidopsis Nudix hydrolase, AtNUDX1, acts to hydrolyze an oxidized deoxyribonucleotide, 8-oxo-7,8-dihydro-2'-deoxyguanosine 5'-triphosphate (8-oxo-dGTP). Here we showed that 8-oxo-dGTP pyrophosphohydrolase activity is not exhibited by any other Arabidopsis Nudix hydrolase. AtNUDX1 acted on an oxidized ribonucleotide, 8-oxo-GTP, with high affinity (K(m) 28.1 microM). In a transcriptional mutational analysis using the lacZ reporter gene, the phenotypic suppression of the lacZ amber mutation in a mutT-deficient Escherichia coli strain caused by the misincorporation of 8-oxo-GTP into the mRNA was significantly diminished by expression of AtNUDX1. These findings suggest that AtNUDX1 prevents transcriptional errors in vivo. A confocal microscopic analysis using a green fluorescent protein (GFP) fusion protein demonstrated that AtNUDX1 is distributed in the cytosol, where the main pool of nucleotides in the cells exists. The level of 8-oxo-guanosine in genomic DNA was significantly increased in knockout nudx1 plants compared with wild-type plants under normal and oxidative stress (3 microM paraquat) conditions. The results obtained here indicate that AtNUDX1 functions in cellular defense against oxidative DNA and RNA damage through the sanitization of their precursor pools in the cytosol in Arabidopsis cells.