The nucleotide sequence of a 6.4 kbp portion of the 10.6 kbp BamHI fragment D contained in the unique short region of the equine herpesvirus type 1 (EHV-1) genome has been determined. Analysis of this sequence revealed five open reading frames (ORFs), four complete and one incomplete, which were encoded by the same sense strand. Comparison of the EHV-1 DNA sequence with that encoding glycoproteins of other alphaherpesviruses has revealed no significant homologies. Comparison at the amino acid level, however, has demonstrated regions of significant sequence similarity between the three complete EHV-1 ORFs 2, 3 and 4, and the herpes simplex virus type 1 (HSV-1) glycoprotein gD encoded by the US6 gene, the HSV-1 glycoprotein gI encoded by the US7 gene and the HSV-1 glycoprotein gE encoded by the US8 gene, respectively. The interrupted ORF 5 was found to display partial homology with the HSV-1 US9-encoded protein, but no homology was found between the protein encoded by ORF 1 and other proteins. The three collinear EHV-1 ORFs encoding putative glycoproteins with homology to the HSV-1 glycoproteins were therefore designated EHV-1 gD, gI and gE, respectively. Moreover, further similarities were found between EHV-1 gD and pseudorabies virus (PRV) gp50, between EHV-1 gI and PRV gp63 and varicellazoster virus (VZV) gpIV, and between EHV-1 gE and PRV gI and VZV gpI. It is concluded that EHV-1, PRV, HSV-1 and VZV encode homologous glycoprotein genes in the small unique components of their genomes and that the genetic organization of these regions is conserved.