The nucleotide sequences of the glycoprotein B (gB) genes of Marek's disease virus (MDV) serotypes 2 and 3 were determined (gB-2 and gB-3, respectively). The genomic locations of these genes coincide with that of the gB gene of serotype 1 MDV (gB-1). Alignment with gB-1 (Ross et al., 1989, J. Gen. Virol. 70, 1789-1804) revealed predicted amino acid identities of 83 and 82% for gB-2 and gB-3, respectively. Excluding the predicted N-terminal signal sequences, 8 of 9 potential N-linked glycosylation sites and all 10 cysteine residues in gB-1 are conserved in both gB-2 and gB-3. In addition, the putative proteolytic cleavage sites for processing of precursors (gp100s) to gp60s and gp49s are conserved among the three gB homologs. Fowlpox virus (FPV) recombinants expressing either the gB-2 or the gB-3 gene were constructed. We detected expression of authentic gB-2 and gB-3 complexes in cells infected with these FPV recombinants. Digestion of immunoprecipitated gB-1 and gB-3 with endoglycosidases revealed that both gp60s are modified by the additions of O-glycans and complex carbohydrates after cleavage of gp100s, while gp100s and gp49s contain only high-mannose carbohydrates. We confirm that the size differences between gB-1 and gB-3 complexes are due to different carbohydrate modifications.