Aphidicolin, a specific inhibitor of cellular DNA polymerase alpha and of viral DNA polymerase, inhibits production of infectious virus and cellular and viral DNA synthesis of human cytomegalovirus (HCMV)-infected cells. On the other hand, 2',3'-dideoxynucleosides, inhibitors of DNA polymerases beta and gamma, do not affect HCMV replication. The data show that the alpha DNA polymerases of either viral or cellular origin are required for viral DNA synthesis, and cannot be substituted by the cellular DNA polymerase beta and gamma.