This report compares the effect of the newly synthesized 1-beta-D-arabinofuranosylthymine 5'-triphosphate with 1-beta-D-arabinofuranosylcytosine 5'-triphosphate on the activity of DNA polymerases from mouse cells and oncornavirus. 1-beta-D-Arabinofuranosylthymine 5'-triphosphate inhibited all the activities of DNA polymerase alpha, beta, and gamma and viral DNA polymerase. The mode of inhibition of 1-beta-D-arabinofuranosylthymine 5'-triphosphate as well as 1-beta-D-arabinofuranosylcytosine 5'-triphosphate was competitive to the deoxynucleoside triphosphate with the same base. The inhibition constant (Ki) and the mode of inhibition of nucleotide incorporation varied with changes in the combination of the inhibitor, substrate(s), and enzyme species.