The acyclic nucleoside phosphonate analogue (S)-1-[3-hydroxy-2-phosphonylmethoxypropyl]cytosine (HPMPC) is a potent and selective inhibitor of human cytomegalovirus (HCMV) replication and DNA synthesis. Unlike 9-(1,3-dihydroxy-2-propoxymethyl)guanine (DHPG), HPMPC inhibits HCMV replication in cell cultures which have been treated with the compound before infection. Upon short-pulse treatment of HCMV-infected cells with HPMPC, a long-lasting antiviral response is obtained. The antiviral activity of HPMPC, unlike the antiviral activity of other cytosine derivatives (i.e. Ara-C, FIAC), is not readily reversed by 2'-deoxycytidine or cytidine. Neutral and alkaline sucrose gradient analysis of HCMV DNA isolated from HPMPC-treated HCMV-infected cell cultures revealed that HPMPC does not cause (detectable) single- or double-strand breakage of HCMV DNA.