Infected-cell protein 4 (ICP4) is the major transcriptional activator of herpes simplex virus (HSV) gene expression during productive infection. ICP0 has broad transactivating activity for all classes of HSV genes as well as cellular genes and genes of heterologous viruses. Together, the transactivating activities of ICP4 and ICP0 are synergistic. ICP27, which alone does not exhibit major transregulatory activity, is able to differentially activate and repress viral gene expression induced by ICP4 and ICP0. Thus, ICP27 plays a modulatory role in viral gene expression. In order to explore the functional relationships among ICP4, ICP0, and ICP27 in the regulation of viral gene expression, we have used indirect immunofluorescence to examine the intracellular localization of ICP4 in cells infected with wild-type virus or with mutant viruses that did not express functional forms of ICP0 or ICP27. Although ICP4 localized to both the nuclei and cytoplasm of cells infected with either the wild-type virus or an ICP0 null mutant virus, this protein was present exclusively in the nuclei of cells infected with an ICP27 null mutant virus, suggesting that ICP27 is able to inhibit the nuclear localization of ICP4 during virus infection. Transient expression assays with pairs of plasmids that express wild-type forms of ICP4 and ICP0 or of ICP4 and ICP27 demonstrated that ICP27 has a significant inhibitory effect on the nuclear localization of ICP4, confirming the observations made with the mutant-virus-infected cells. By using a plasmid expressing wild-type ICP4 and a series of ICP27 mutant plasmids in transient expression assays, the C-terminal half of ICP27 was shown to be required for its inhibitory effect on the nuclear localization of ICP4. In similar studies using a series of ICP4 mutant plasmids, the region of ICP4 responsive to wild-type ICP27 was mapped to the C-terminal portion of the molecule between amino acid residues 820 and 1029. The level of expression of ICP27 was shown to have a significant effect on the intracellular localization of ICP4 in transient assays. These findings are consistent with previous studies in which ICP27 was shown to have an inhibitory effect on the nuclear localization of ICP0 (Z. Zhu, W. Cai, and P. A. Schaffer J. Virol. 68:3027-3040, 1994). Thus, ICP27 has a significant inhibitory effect on the ability of the two major HSV type 1 (HSV-1) regulatory proteins to localize to the nucleus. Collectively, these findings indicate that cooperative regulation of HSV-1 gene expression may well involve intracellular compartmental constraints.