Infection of secondary mouse fibroblast cultures with human cytomegalovirus (HCMV) led to the production of viral antigens in the absence of detectable virus replication. Antigen production was not dependent on viral DNA synthesis since it was not inhibited by cytosine arabinoside. Beginning at 15 days post-infection, viral-specific antigens were no longer observable in infected cultures, but could be induced by iododeoxyuridine (IUDR) treatment. Such cultures carry HCMV genetic information in a latent, unexpressed, state. Fusion of latently-infected mouse cells with human fibroblasts also induced viral-specific antigens, but no detectable virus replication, in the resulting heterokaryons. However, upon IUDR treatment of heterokaryons, or of the mouse cells prior to fusion, antigens characteristic of the late stages of virus replication appeared, and infectious CMV could be detected. Thus, it appears that the non-permissive (mouse) cell genome is dominant, and continues to regulate viral gene expression in heterokaryons formed with permissive (human) cells.