Five temperature‐sensitive mutants (tsm9, tsm13, tsm20, tsm22, tsm30) of murine cytomegalovirus have been shown previously not to produce infectious virus in mice. In the present study, the stage at which these mutants are blocked in their replication in vitro was examined by transcriptional analysis of 4 temporally regulated marker genes (IE‐1, E‐1, gB and gH) using a semi‐quantitative reverse transcription polymerase chain reaction (RT‐PCR) coupled with an electron microscopic analysis of infected cells incubated at permissive (33°C) and non‐permissive (39 and/or 40°C) temperatures. Replication of tsm13 appeared to be blocked at a late phase of replication after capsid formation while the block appeared to be as early as the immediate‐early phase in tsm22‐ infected cells. In contrast, mutants tsm9, tsm20 and tsm30 were blocked at a maturation step, probably of capsid formation, as gene transcription of all 4 marker genes occurred, albeit at reduced level, at 39 and 40°C but no capsids or virions were produced at 40°C. Replication and transcription of mutants tsm13, tsm20 and tsm30 were also examined in infected mice. Mutant tsm13 showed no gene expression or infectious virus while mutants tsm20 and tsm30 produced no infectious virus from days 3–60 post infection, except unusually for a low titre of tsm30 (2.3 x 10³ pfu/ml) in salivary glands 21 days post infection. Gene transcription of all 4 marker genes was observed in one or more tissues (salivary glands, spleen, kidneys, liver, thymus, heart, lungs) at one or more time points (3, 7, 10, 14, 21 days post‐infection) with both mutants. Mice became infected latently with tsm20 but not tsm30, and mice previously infected with tsm20 or tsm30 were protected against a sub‐lethal challenge with virulent parental virus; tsm30 also protected against a lethal challenge. This suggests that these two mutants may be good model vaccines for further studies on the mechanism of protection induced and for identification of the ts genes. J. Med. Virol. 62:127–139, 2000. © 2000 Wiley‐Liss, Inc.