Cytomegalovirus (CMV) DNA polymerase has immunologic specificity in relation to the virus specific polymerases of other herpesviruses. During the early phase of CMV infection in vitro, the virus DNA polymerase is rapidly induced. Due to the lack of virus specific thymidine kinase, cytomegalovirus is resistant to nucleosides which require herpesvirus thymidine kinases for activation. Cytomegalovirus DNA polymerase is therefore the known possible target for antiviral drugs. Several pyrophosphate analogs have been assayed for their inhibitory effects on this enzyme. The most active compound is phosphonoformate (PFA). PFA also effectively inhibits other partially purified herpesvirus DNA polymerases as well as the multiplication of all human herpesviruses, at concentrations which do not affect cellular DNA polymerases or normal cell growth. The mechanism of inhibition is a noncompetitive inhibition of CMV DNA polymerase activity with respect to the four deoxyribonucleoside triphosphates, and an uncompetitive inhibition with respect to the template used. In cell culture, PFA inhibits the formation of late CMV polypeptides, but not the synthesis of early CMV polypeptides. The CMV specific polymerase persists in the presence of PFA, as measured by immunological methods, and enzyme activity can be demonstrated after removal of PFA. The inhibition of CMV replication is reversible even after long exposure to PFA. Our interpretation is that the CMV genome is highly resistant to the cellular metabolism also in non-producing cells. A new rapid CMV neutralization test was established, based on the appearance of early CMV-induced antigens.