Phosphonoacetic acid (PAA) inhibits the replication of human herpesvirus-6 (HHV-6) in mononuclear cells from cord bloods which are susceptible for natural HHV-6 infection in humans. Nuclear extracts of uninfected or HHV-6-infected mononuclear cells were applied to phosphocellulose column chromatography, and DNA polymerase activity was measured with or without the addition of 100 mM ammonium sulfate. The major DNA polymerase activities eluted at 0.47 M KCl were suppressed in both uninfected and HHV-6 infected cells by the addition of 100 mM ammonium sulfate. DNA polymerase activity eluted at 0.47 M KCl was observed only from HHV-6-infected cells; it was enhanced by 100 mM ammonium sulfate and neutralized with immune serum. DNA polymerase activity eluted at 0.73 M KCl was determined to be HHV-6 specific and had the properties of a typical herpesvirus-induced DNA polymerase. PAA inhibited HHV-6-specific DNA polymerase activity.