We have previously identified distinct nuclear and mitochondrial isoforms of dUTPase in human cells, reporting the cDNA sequence of the nuclear isoform (DUT-N). We now report a cDNA corresponding to the mitochondrial isoform (DUT- dUTPase (EC 3.6.1.23) catalyzes the hydrolysis of dUTP to dUMP and pyrophosphate, simultaneously removing dUTP from the DNA biosynthetic pathway and providing substrate (dUMP) for the de novo synthesis of thymidylate (1). The importance of the dUTPase function in prokaryotic, eukaryotic, and viral systems has been firmly established in recent years. dUTPase is essential for viability in Escherichia coli and Saccharomyces cerevisiae and was shown to be required for efficient DNA replication in several viral systems (2-5). The importance of the dUTPase function in the human system has been further distinguished by its importance in anti-thymidylate chemotherapy.MThymidylate metabolism has been an important target for the development of widely utilized chemotherapeutic agents such as 5-fluorouracil, fluorodeoxyuridine, and ZD1694 that are used in the treatment of breast and gastrointestinal tumors. The enzymatic target of these compounds, thymidylate synthase (TS), 1 generates dTMP from dUMP and a folate cofactor. Investigations by several laboratories suggest that the basis of cytotoxicity caused by inhibition of de novo thymidylate metabolism may be the accumulation of excessive dUTP pools (6 -9). TS inhibition induces a dramatic elevation of dUTP pools resulting in chronic dUMP misincorporation into DNA during replication and repair, leading to DNA fragmentation and cell death (7). Work published by Curtin et al. (7) demonstrates a significant correlation between intracellular dUTP levels and the magnitude of DNA damage resulting from TS inhibition. dUTPase (the major regulator of dUTP pools in humans) plays a protective role by limiting dUTP accumulation in the cell and countering the cytotoxic effect of TS inhibition. Recently, direct evidence supporting this role was demonstrated. Overexpression of the E. coli dUTPase in HT29 human colorectal tumor cells resulted in the induction of resistance to the TS inhibitor fluorodeoxyuridine (10). These studies provide substantial evidence suggesting that levels of the dUTPase enzyme may be a critical factor in determining fluorodeoxyuridine toxicity in certain cancer types. The essential nature of dUTPase during