The herpes simplex virus (HSV) type 1 immediate-early protein ICP4 represses transcription of its own gene and possibly that of other immediate-early genes. In the present study, we analyzed the role that an ICP4 binding site present in two HSV-1 promoters plays in the level and kinetics of expression during HSV-1 infection. Wild-type and mutant forms of the IE-3 (ICP4) promoter and the latency associated promoter (LAP) were fused to the thymidine kinase (tk) coding sequences and transferred to the genome of an ICP4-deficient virus. Promoter mutants were constructed to assess the effect of the ICP4 binding site in the presence and absence of defined binding sites for cellular and other viral factors in the promoters. The activities of the promoter constructs were inferred from the level of tk mRNA seen following viral infection in the absence of ICP4, in Vero cells and in the presence of ICP4 in ICP4 expressing E5 cells. Kinetics of expression and the dependence on DNA synthesis for expression were examined following infection of E5 cells. In the presence of the ICP4 binding site in LAP and in IE3 promoters lacking TAATGARAT motifs, expression was maximal late after infection and was greatly reduced when DNA synthesis was inhibited. When the ICP4 binding site was removed, both LAP and the IE3 construct retaining an Sp1 site were more abundantly expressed and exhibited kinetics of expression indistinguishable from that of the tk promoter. In vitro, ICP4 repressed LAP transcription mediated by the general transcription factors and upstream activating proteins. Deletion of the ICP4 binding site not only relieved repression, but in the presence of USF activity, ICP4 further induced LAP transcription. The results of these experiments suggest a role for the repressor activity of ICP4 in the temporal regulation of HSV-1 transcription.