To characterize the formation of replication compartments in human cytomegalovirus-infected cells, and to determine the fate of newly synthesized DNA, we localized viral replication proteins and DNA synthesis at early and late times during infection. As expected, ppUL57 (single-stranded DNA binding protein) and ppUL44 (DNA polymerase processivity factor) both localized to replication compartments beginning at 48 hpi. BrdU was incorporated into viral DNA in these compartments that was found to mature into progeny virus based on our ability to chase the label into the cytoplasm and out of the cell over the ensuing 72-h period. Although the pattern of BrdU incorporation at early times (20 or 24 hpi) was punctate, and distinct from the replication compartment that formed later during infection, viral DNA synthesized at this time also matured into progeny virus during a chase. Interestingly, sites of ppUL57 localization did not overlap completely with sites of BrdU incorporation at early times. Products from the UL112-113 gene localized to subnuclear regions by 6 hpi, earlier than ppUL57. Between 12 and 24 hpi, both ppUL57 and ppUL44 joined UL112-113 gene products at sites that subsequently developed into replication compartments. When infection was carried out in the presence of phosphonoformate or ganciclovir, replication compartment formation was blocked. A viral mutant deficient in uracil DNA glycosidase, previously shown to exhibit a delay in the initial phase of DNA replication, also exhibited delayed formation of replication compartments. These results raise the possibility that subnuclear sites defined by UL112-113 localization orchestrate the assembly of the CMV replication compartment and implicate punctate sites of BrdU incorporation as sites of early viral DNA replication that precedes the formation of the replication compartment.