5-Methoxymethyl-2′-deoxycytidine (MMdCyd) is a selective anti-herpes agent that is dependent upon initial activation by herpes simplex virus (HSV)-induced deoxythymidine/deoxycytidine kinase. 5-Methoxymethyl-2′-deoxycytidine triphosphate (MMdCTP) was synthesized. The nature of the interaction of MMdCTP and dCTP with the DNA polymerase of Escherichia coli, HSV-1 and human DNA polymerase α was determined using specific and optimized assay conditions for each enzyme. MMdCTP was a better substrate for HSV-1 DNA polymerase compared to dCTP. At a nucleotide concentration of 10 μm MMdCTP utilization was 130% that of an equimolar concentration of dCTP. Under similar conditions, human DNA polymerase α utilized MMdCTP about as efficiently as dCTP. E. coli DNA polymerase I preferentially utilized dCTP. The IC50 values of MMdCTP were 8 × 10−7 m and 29 × 10−7 m for HSV-1 and human α DNA polymerase, respectively. MMdCTP is a competitive inhibitor of HSV-1 DNA polymerase with respect to dCTP incorporation ( Ki = 3.8 × 10−7 m). Preferential utilization of MMdCTP and its eventual incorporation into HSV DNA seems to account for the antiviral action of MMdCyd.