Many cytomegalovirus (CMV) infections result from activation of virus previously latent in the host. Murine models of latent CMV infection have been developed in which latent virus can be activated in vivo by immunosuppression or by coculture of splenic lymphocytes in vitro. In the present study, latent murine CMV (MCMV) could be activated from lymphocytes of mice regardless of genetic strain, age at time of virus inoculation, or use of syngeneic or allogeneic fibroblasts for coculture. After intraperitoneal inoculation, virulent virus was activated from lymphocytes more often than attenuated MCMV (69% vs. 20% of lymphocyte cocultures were positive, respectively). Latent MCMV was not detected in lymphocytes after subcutaneous inoculation of weanling mice but could be activated from mice infected subcutaneously as newborns. The absence of latent infection in the lymphocytes of the mice that had been inoculated as weanlings was due to lack of virus replication in the spleen during acute infection.