Herpes simplex virus mutant strains that fail to encode a functional ICP27 polypeptide show a reduction in their ability to replicate viral DNA compared to wild-type virus. To define the nature of this defect, we examined the formation of viral DNA replication structures in the nucleus of cells infected with these mutant viruses. In cells infected with wild type HSV, viral DNA replication occurs in replication compartments. If viral DNA replication is blocked during infection, a subset of these proteins are found in a punctate staining pattern termed prereplicative sites. ICP8, an essential DNA replication protein, is required for formation of these structures and for localization of polymerase and other proteins to these structures. To study the assembly of replication proteins in cells infected with ICP27 defective mutants, we examined the intranuclear localization of ICP8. Our results suggest that assembly of replication proteins into active sites is defective in a majority of cells infected with mutant virus encoding altered ICP27 proteins. Furthermore, this defect in assembly occurs at an early step in localization of replication proteins, possibly due to an observed change in the conformation of ICP8. Last, only actively dividing cells at a certain stage of the cell cycle are able to form replication compartments when infected with ICP27 defective mutants. The residual DNA synthesis seen with these mutants occurs in a defined subpopulation of infected cells. Thus, ICP27 may promote the proper folding and/or localization of ICP8.