Herpes simplex virus types 1 and 2 (HSV-1 and HSV-2), pseudorabies virus (PRV), varicella-zoster virus (VZV), and equine herpesvirus 1 (EHV-1) are all classified as Alphaherpesvirinae. Each of these five viruses encodes an essential immediate-early (IE) regulatory protein referred to as HSV-1 ICP4, HSV-2 ICP4, PRV IE180, VZV ORF62 protein, and EHV-1 IE1, respectively. These five proteins share extensive homology with each other in domains referred to as regions 2 and 4. The HSV-1 ICP4 region 2 domain contains residues that are required for the DNA-binding capability of ICP4. In this report, we describe the expression of region 2 domains from the ICP4, IE180, and ORF62 genes as fusion proteins in Escherichia coli. DNA-binding assays revealed that each of these region 2 fusion proteins binds to a sequence that overlaps the transcription start site in the promoter for the gene encoding the corresponding protein. Each of the sites with high affinity for one or more of these fusion proteins contains the sequence 5'-ATCGT-3'. This sequence spans the mRNA cap site in the HSV-2 ICP4 gene promoter and is immediately upstream from the transcription start site in the EHV-1 IE1 gene. These results suggest that formation of a specific complex between an IE protein and its own gene promoter may be a common mechanism used by Alphaherpesvirinae to autoregulate transcription of an essential IE gene.