DNA polymerase is one of the most important target molecules of antitumor agents, especially for antimetabolite nucleosides that include 1-β-D-arabinofuranosylcytosine (araC) and 2-deoxy-2,2-difluorocytidine (gemcitabine). There are several subtypes of mammalian DNA polymerases and their localization and function have been clarified. DNA polymerase α, δ and ε have been implicated to be responsible for DNA replication, whereas DNA polymerase β, δ and e have been suggested to work in DNA repair. DNA polymerase γ is encoded in the nucleus but localizes in the mitochondria, and is responsible for the mitochondrial DNA replication. Recently, several antiviral nucleoside analogs were reported to inhibit DNA polymerase g after intracellular phosphorylation and cause severe chronic toxicity. 1-(2-Deoxy-2-fluoro-4-thio-β-D-arabinofuranosyl)cytosine (4-thio-FAC), an antimetabolite similar to araC and gemcitabine, is recently shown by us to be a very promising agent because of its potent antitumor activity by oral administration to mice. We tested for the inhibitory activities of the triphosphates of 4-thio-FAC and gemcitabine against DNA polymerase α, β and γ. The triphosphates of 4-thio-FAC (4-thio-FACTP) exhibited the potent inhibitory action against DNA polymerase a, whereas it showed moderate inhibition against DNA polymerase β and little inhibition against DNA polymerase γ. The triphosphate of gemcitabine (dFdCTP) did not show potent inhibition against these three DNA polymerases. Thus, the effect on ribonucleotide reductase was suggested to be more responsible for the antitumor action of gemcitabine. The differences in the mechanisms of action against DNA polymerases between these drugs and other nucleosides were also discussed.