Cidofovir [CDV,(S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine, HPMPC] is an acyclic cytosine nucleoside phosphonate analog with potent in vitro and in vivo activity against a broad spectrum of herpesviruses. CDV diphosphate (CDVpp), the putative antiviral metabolite of CDV, is a competitive inhibitor of dCTP and an alternate substrate for human cytomegalovirus (HCMV) DNA polymerase. HCMV DNA polymerase used a synthetic DNA primer-template with a Km value of 90 +/- 8 nM and incorporated dCTP approximately 42 times more efficiently than CDVpp. HCMV DNA polymerase also utilized a synthetic DNA primer containing a single molecule of CDV at the 3'-terminus. The Km value for this DNA primer-template was 165 +/- 42 nM and incorporation of dCTP was approximately 17 times more efficient than that of CDVpp. The slower rate of incorporation of CDVpp was due mostly to the higher Km value of CDVpp toward the enzyme-primer-template complexes. These data demonstrate that incorporation of a single CDV into DNA by HCMV DNA polymerase does not lead to chain termination.