The DNA sequence of a 5.5 kbp EcoRI fragment located in the short unique region (Us) of the 'highly oncogenic' strain RB1B of Marek's disease virus (MDV) was determined. The sequence contained six open reading frames (ORFs), four of which were homologous to proteins mapping in the Us region of herpes simplex virus type 1 (HSV-1). These include the homologues of HSV-1 protein kinase, glycoprotein D (gD), glycoprotein I (gI) and US2 which is of unknown function. The MDV ORFs had a marked bias for A or T in the third codon position and analysis of the dinucleotide frequencies showed a marginal deficit in ApG/CpT but no overall deviation of CpG from random expectations. Comparison of genes in the Us region of MDV to he~esvirus proteins confirmed and extended our previous observation that MDV is more closely related to alphaherpesviruses than to gammaherpesviruses. We also showed that MDV possessed a homologue of HSV-1 gD which is lacking in varicellazoster virus (VZV) but that MDV probably lacked homologues of US4 and US5 of HSV-1. These results show that in contrast to the genes in the long unique region which were grossly collinear in HSV, VZV and MDV, those mapping in Us show greater diversity.Marek's disease virus (MDV) DNA is a linear, 175 kbp double-stranded molecule consisting of unique long (UL) and unique short (Us) segments flanked by inverted repeats (IRs) (Cebrian et al., 1982). Recently, Buckmaster et al. (1988) identified 35 MDV genes by comparing the translated sequence of random fragments of MDV DNA with the amino acid sequences of known herpesvirus proteins. Most of the genes identified mapped in the UL region and were coUinear with genes of the alphaherpesviruses herpes simplex virus (HSV) and varicella-zoster virus (VZV). Moreover, the random sequencing study showed that MDV was more closely related to alphaherpesviruses than to beta-and gammaherpesviruses on the basis of amino acid sequence conservation. Genes of MDV that have been sequenced fully so far map in UL (Coussens & Velicer, 1988;Scott et al., 1989). In this paper we report on the DNA sequence and organization of predicted open reading frames (ORFs) in approximately half of the Us segment of MDV strain RB1B (Schat et al., 1982). We were particularly interested in determining whether MDV had a homologue of the HSV glycoprotein D (gD) and the nature and arrangement of genes adjacent to gD.Initially, random fragments of MDV DNA derived from a 23 kbp BamHI A fragment of the GA strain of MDV (Fukuchi et al., 1984) containing part of Us and most of the IRs (Fig. 1) were sequenced and the TRL