The gene encoding the precursor polypeptide of the Marek's disease herpesvirus (MDHV) secretory glycoprotein gp57-65 (formerly identified as A antigen) has been sequenced. Previous results had localized the gene to a 4.6-kilobase (kb) segment of the BamHI B fragment in the unique long region of the MDHV genome. S1 nuclease protection experiments were used to more precisely locate the 5' initiation and approximate 3' termination points of the approximately 1.8-kb MDHV gp57-65 mRNA within this segment. These results indicated that the entire MDHV gp57-65 coding sequence is contained within a 2.35-kb PvuII-EcoRI fragment, with the direction of transcription from PvuII to EcoRI (5' to 3'). Nucleotide sequence analysis of this region revealed a single open reading frame of 1,515 base pairs. The MDHV gp57-65 coding sequence has an overall guanosine-plus-cytosine content of 41%. Translation of the single open reading frame would produce a polypeptide of 505 amino acids, with a calculated molecular weight of 56,805. The putative gp57-65 precursor polypeptide contains features common to many glycoproteins. These include a hydrophobic amino-terminal region (amino acids 1 to 27) that may function as a signal peptide and nine potential N-linked glycosylation sites (Asn-X-Ser/Thr). These two features, predicted from nucleotide sequence data, are consistent with the published data showing that gp57-65 has a signal peptide and N-linked glycosylation (R. J. Isfort, R. A. Stringer, H.-J. Kung, and L. F. Velicer, J. Virol. 57:464-474, 1986). The predicted sequence indicates that overall the polypeptide is relatively hydrophobic, with a possible 18-residue carboxyl-terminal membrane anchor sequence. This sequence appears to be less prominent than those commonly found in integral membrane glycoproteins. The lack of a strong hydrophobic anchor sequence may help to explain the predominantly secretory nature of MDHV gp57-65.