Phosphonylmethoxyalkylpurines and -pyrimidines, particularly (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine (HPMPC) and (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine (HPMPA) and their cyclic forms (cHPMPC, cHPMPA) and the 3-deaza analogue of HPMPA (HPMPc3A) are selective and potent inhibitors of human cytomegalovirus (CMV) replication in vitro. Their anti-CMV activity has been monitored by flow cytometry and DNA hybridization. The anti-CMV agent ganciclovir [9-(1,3-dihydroxy-2-propoxymethyl)guanine (DHPG)] was included as a reference compound. From the flow cytometric assays, HPMPC and HPMPA were the most active compounds, with an EC50 (50% effective concentration) of approximately 0.6 microM. The EC50 values obtained by DNA hybridization ranged from 0.05 microM (HPMPC) to 0.74 microM (DHPG). Selectivity indexes, calculated as the ratio of the 50% inhibitory concentration (CC50) for cell growth or [methyl-3H]-thymidine incorporation to the EC50 for virus replication were highest for HPMPC and its cyclic derivative (cHPMPC). The flow cytometry and DNA hybridization assays thus confirm the results obtained by the classic plaque assay. They allow a quantitative estimation of the anti-CMV potency of the test compounds.