Herpes simplex virus type 1 (HSV-1) infection results in the disruption of ND10 (also called nuclear bodies, PODs, or PML-associated bodies), which are nuclear matrix domains of unknown function present in mammalian cells. After ND10 disruption, viral transcription and DNA replication occur in globular nuclear domains called replication compartments. In this report we define four stages of infection by using antibodies to ICP8 (also called SSB and UL29) and the ND10 antigen PML. Immediately after infection, cells contain intact ND10 as detected by staining for PMLs (stage I); within 1 hour, however, ND10 are disrupted and cells begin to exhibit diffuse staining for the major viral DNA binding protein, ICP8 (stage II). After all ND10 have been disrupted, foci which resemble but are not equivalent to ND10 appear, containing both PML and ICP8 (stage III). Cells infected with mutants defective in the helicase-primase or origin binding protein are unable to form stage III foci. Cells infected with a mutant that is null for the polymerase catalytic subunit, however, form stage III-like ICP8 foci which do not contain PML. Thus, stage III foci recruit the cellular PML protein in the presence but not the absence of HSV polymerase. PML was recruited to stage III foci in some but not all cells infected with a mutant defective in the polymerase accessory protein, UL42. Thus, UL42 is not required for the recruitment of PML to viral foci. In wild-type infection, stage III cells are quickly replaced by cells containing replication compartments (stage IV). PML and ICP8 staining are both observed within replication compartments, indicating a potential role for PML in HSV-1 replication. Models for the role of ND10 proteins in the formation of replication compartments are discussed.