The nucleotide sequence of the gene encoding glycoprotein B (gB) of rhesus cytomegalovirus (RhCMV) was determined and the protein characterized. The open reading frame of gB encoded a protein of 854 amino acids with 60% identity and 75% similarity at the amino acid level to human cytomegalovirus (HCMV) gB. Cysteine residues in the extraluminal part of the protein are perfectly conserved. Out of the 16 potential N-linked glycosylation sites present in HCMV gB, 15 are conserved in RhCMV gB. Immunoblot analyses with antisera detected three bands of 150 kDa, 90-110 kDa and 55 kDa representing the full-length gB as well as the proteolytic cleavage products. Cross-reactivity and cross-neutralization of a number of HCMV gB-specific monoclonal antibodies with RhCMV gB indicated sharing of immunogenic epitopes between the two molecules. The RhCMV gB regions corresponding to antigenic domains AD-1, 2 and 3 of HCMV gB were immunogenic during natural RhCMV infection with the AD-1 region being the immunodominant domain. The data indicate that RhCMV might represent a useful model to investigate pathogenesis and immune surveillance of cytomegaloviruses.