DNA replication during human or simian cytomegalovirus (CMV) infection has been shown to be under control of a replicator region referred to as oriLyt. The murine CMV oriLyt has been mapped to a region of the genome located upstream of the gene encoding the herpesvirus-conserved single-stranded DNA binding protein, analogous to human and simian CMV oriLyts. A minimal oriLyt of approximately 1.7 kbp has been identified using a transient replication system. Like occurs with human and simian CMV counterparts, addition of flanking sequences to this minimal origin-stimulated replication efficiency. Analysis of the DNA sequence in this region shows that murine CMV oriLyt is complex and exhibits an asymmetric distribution of nucleotides as well as many repeat sequence elements, including distinct AT- and GC-rich regions and region with arrays of closely spaced direct repeats. Despite similarities in organization of all three CMV oriLyts, no sequence identity and only limited DNA sequence similarity was detectable. Consistent with this sequence divergence, the human and murine CMV oriLyts were unable to substitute for one another in transient replication assays.