The mammalian endonuclease III homolog NTH1 is a DNA glycosylase/AP lyase that recognizes oxidized pyrimidine bases. Here, we compared the intracellular localization of human and mouse NTH1 and analyzed their sorting signals by examining expression of enhanced green fluorescent protein (EGFP)-tagged NTH1 protein. Full-length hNTH1 was sorted exclusively into nuclei. Deletion analysis showed that two basic amino acid clusters, which constitute the nuclear localization signal (NLS), are essential for nuclear sorting. Moreover, disruption of the NLS by deletion or substitution of arginine residue(s) altered the localization of the protein to mitochondria. In contrast, most mNTH1 molecules were sorted into mitochondria, with a relatively small amount localized in nuclei. Deletion analysis indicated that the mitochondrial targeting sequence of mNTH1 is contained within the N-terminal 38 amino acids. Alignment of the N-terminal sequence of human and mouse NTH1 showed that mNTH1 lacks a basic amino acid cluster corresponding to one of the NLS sequences found in hNTH1. Nuclear localization of mNTH1 was increased when this NLS sequence was added to mNTH1 through the addition of appropriate amino acids. The fact that transcription of the hNTH1 gene is initiated at multiple sites indicated that three isoforms of hNTH1 protein are translated using different initiation codons. However, no difference in intracellular localization was observed among three isoforms of hNTH1 with different N-terminal sequences.