5-Methoxymethyl-2′-deoxyuridine (MMdUrd) is a selective anti-herpes agent that is dependent upon initial phosphorylation by Herpes simplex virus-induced deoxythymidine kinase. In order to determine its mechanism of action, MMdUrd was converted to the 5′-triphosphate (MMdUTP) and the nature of interaction of MMdUTP and dTTP with DNA polymerase of E. coli, HSV-1, and human α was investigated. The order of utilization of deoxyuridine analogues by bacterial and HSV-1 DNA polymerases for DNA synthesis was: dTTP > MMdUTP. In contrast, 5-methoxymethyl-2′-deoxycytidine-5′-triphosphate (MMdCTP) was a better substrate for HSV DNA polymerase compared to dCTP. MMdUTP is a competitive inhibitor of HSV-1 DNA polymerase with respect to dTTP incorporation (Ki= 2.9 × 10−6M). The IC50values of MMdUTP for both HSV and human αDNA polymerases were 4.5 × 10−6M. These data suggest that the selective activity of MMdUrd is due to its preferential phosphorylation by viral thymidine kinase and not at the DNA polymerase level. These results may also account for the difference in anti-HSV activity between MMdUrd and its deoxycytidine analogue.