Studies with 6 ts mutants of mouse cytomegalovirus indicated that mutants tsm1, tsm2, tsm3, and tsm6, like wild-type (wt) virus, produced acute infection in mice, became latent, and were reactivated as infectious virus immunosuppression. Using PCR, all five viruses expressed immediate-early (IE)-1, early (E)-1, and late (L, gB) genes during acute infection in all tissues examined (salivary glands, lung, spleen, liver, kidney, and heart). DNA was present in most tissues during latent infection with all five viruses, but transcription was restricted to the IE-1 gene in the salivary glands of wt infected mice only, suggesting true molecular latency rather than low level virus persistence. Similarly, mutant tsm5 expressed all three genes following primary inoculation. Although no detectable virus was produced, tsm5 subsequently entered the latent state as evidenced by DNA detection without RNA transcription indicating that productive infection is not required to initiate latency. This mutant also failed to reactivate from latency, although all three marker genes were expressed in most tissues. In contrast, tsm4 expressed all three marker genes and produced infectious virus during acute infection, then became latent. However, upon immunosuppression to reactivate tsm4, IE-1 and E-1 transcription occurred but neither gB transcription nor infectious virus was detectable in salivary glands, lung, spleen, liver, kidney, heart, or blood. The significance of this with regard to reactivation from latency is discussed.