A number of previous studies have implied that three herpes simplex virus-encoded nuclear transactivator proteins, IE175 (ICP4), IE110 (ICP0), and IE63 (ICP27), may cooperate in transcriptional and posttranscriptional stimulation of viral gene expression. Using double-label immunofluorescence assays (IFA) in transient expression assays, we have examined the intracellular localization of these three proteins in DNA-transfected cells. The IE110 protein on its own forms spherical punctate domains within the nucleus, whereas the IE175 and IE63 proteins alone give uniform and speckled diffuse patterns, respectively. In infected cells, the IE110 punctate granules have been shown to correspond to novel preexisting subnuclear structures referred to as ND10 domains or PODs that contain a variety of cellular proteins, including SP100 and the PML proto-oncogene product. Cotransfection experiments with wild-type nuclear forms of both IE175 and IE110 provided direct evidence for partial redistribution of IE175 into the same punctate granules that contained IE110. Surprisingly, nuclear forms of IE110 were found to move a cytoplasmic form of IE175 into nuclear punctate structures, and a cytoplasmic form of IE110 was able to retain nuclear forms of IE175 in cytoplasmic punctate structures. Therefore, the punctate characteristic of IE110 appeared to both dominate the interactions and override the normal nuclear localization signals. The domains responsible for the interaction mapped to between codons 518 and 768 in 1E110 and to between codons 835 and 1029 in IE175. Importantly, a truncated nuclear form of the 1,298-amino-acid IE175 protein, which lacked the C-terminal domain beyond codon 834, was found to be excluded from the IE110 punctate granules. Cotransfection of nuclear or cytoplasmic IE110 with a truncated nuclear form of IE63 also led to partial redistribution of IE63 into either nuclear or cytoplasmic punctate granules containing IE110. Both the IE63-IE110 and IE175-IE110 colocalization interactions were demonstrated in Vero cells but not in 293 cells. Consequently, they differ from IE110 self-interactions, which correlate with in vitro dimerization and occur efficiently in both cell types. These interactions may help to explain the altered promoter target specificity and synergism observed when IE175 is cotransfected with IE110 in transactivation studies.