Transcription of the major immediate-early (MIE) genes of cytomegaloviruses (CMV) is driven by a strong promoter-enhancer (MIEPE) complex. Transactivator proteins encoded by these MIE genes are essential for productive infection. Accordingly, the MIEPE is a crucial control point, and its regulation by activators and repressors is pertinent to virus replication. Since the MIEPE contains multiple regulatory elements, it was reasonable to assume that specific sequence motifs are irreplaceable for specifying the cell-type tropism and replication pattern. Recent work on murine CMV infectivity (A. Angulo, M. Messerle, U. H. Koszinowski, and P. Ghazal, J. Virol. 72:8502-8509, 1998) has documented the proposed enhancing function of the enhancer in that its resection or its replacement by a nonregulatory stuffer sequence resulted in a significant reduction of infectivity, even though replication competence was maintained by a basal activity of the spared authentic MIE promoter. Notably, full capacity for productive in vitro infection of fibroblasts was restored in recombinant viruses by the human CMV enhancer. Using two-color in situ hybridization with MIEPE-specific polynucleotide probes, we demonstrated that a murine CMV recombinant in which the complete murine CMV MIEPE is replaced by the paralogous human CMV core promoter and enhancer (recombinant virus mCMVhMIEPE) retained the potential to replicate in vivo in all tissues relevant to CMV disease. Notably, mCMVhMIEPE was also found to replicate in the liver, a site at which transgenic hCMV MIEPE is silenced. We conclude that productive in vivo infection with murine CMV does not strictly depend on a MIEPE type-specific regulation.