Murine monoclonal antibodies and polyvalent monospecific antisera reactive with an abundant 68,000 Mr (p68) human cytomegalovirus (CMV) virion protein were used to characterize this protein within CMV-infected cells. The protein was found to partition within the nucleus and cytoplasm of infected cells. Pulse-chase analysis indicated the p68 was degraded into three proteins of 52,000, 51,000 and 50,000 Mr which were found only within infected cells. Both cellular forms as well as the virion p68 were phosphorylated but non-glycosylated. The p68 was synthesized shortly after infection and in the presence of cytosine arabinoside, an inhibitor of viral DNA replication. Studies with monospecific antisera and a panel of monoclonal antibodies specific for the p68 suggested that this protein was not expressed on the surface of infectious virions or infected cells.