The replication of murine cytomegalovirus strain Smith in murine embryonic fibroblasts was investigated at immediate early, early, and late times after infection. Cloned subgenomic HindIII fragments of murine cytomegalovirus DNA served to define the regions of transcription. At immediate early times viral RNA classes ranging in size from 5.1 to 1.05 kilobases (kb) were transcribed mainly from the fragments HindIII-K and -L, whereas low levels of transcription were detected from the two termini HindIII-E and HindIII-N. A characteristic pattern of proteins could be translated from immediate early RNA in vitro. At early and late times after infection transcription from all HindIII fragments occurred, but different patterns of transcripts and proteins could be identified. Inhibitors of DNA synthesis induced differences in the late transcription pattern, located in the HindIII-F fragment. The coding region for abundant immediate early transcription could be located at between 0.769 and 0.817 map units. A plasmic clone containing the main part (0.769 to 0.815 map units) of this region was constructed. This region coded for six polyadenylated immediate early RNA species of 5.1, 2.75, 2.0, 1.75, 1.65, and 1.05 kb in size. Only the 1.75-kb RNA originated entirely from the HindIII-L fragment. The 5.1- and 2.75-kb RNA species were encoded by both the HindIII-L and HindIII-K fragments, and the 2.0-, 1.65-, and 1.05-kb RNA species were entirely transcribed within HindIII-K.