(S)-9-(3-Hydroxy-2-phosphonylmethoxypropyl)adenine [(S)-HPMPA], (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)-2,6-diaminopurine [(S)-HPMPDAP], cyclic (S)-HPMPA [(S)-cHPMPA], 9-(2-phosphonylmethoxyethoxyethyl)-2,6-diaminopurine (PMEDAP), and (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine [(S)-HPMPC] were examined for their inhibitory effects on Epstein-Barr virus (EBV) replication. The 50% effective concentrations for inhibition of viral DNA replication were 0.16, 0.03, 2.0, 1.5, and 0.08 microM for PMEDAP, (S)-HPMPC, (S)-HPMPDAP, (S)-cHPMPA, and (S)-HPMPA, respectively. The relative efficacies based on the in vitro therapeutic index was (S)-HPMPC (5,000) greater than PMEDAP (1,000) = (S)-HPMPA (1,000) greater than (S)-cHPMPA (136) greater than (S)-HPMPDAP (78). Certain ratios of combinations of (S)-HPMPC with 3'-azido-3'-deoxythymidine produced a synergistic inhibitory effect on EBV genome replication, but others exhibited an antagonistic effect. These results indicate that this series of acyclic nucleoside phosphonate analogs, and in particular (S)-HPMPC, are potent and selective anti-EBV agents in vitro.