Rhesus cytomegalovirus (RhCMV) infection of rhesus macaques is an important model to investigate critical issues of cytomegalovirus biology. To better understand host immunological responses to viral glycoproteins, the glycoprotein B (gB) gene of RhCMV was molecularly cloned, sequenced and characterized. Transcription analysis revealed that RhCMV gB was transcribed as a late gene. The RhCMV gB gene encoded a predicted protein of 854 amino acids that was 60% identical/75% similar to the human CMV (HCMV) gB protein. The region of HCMV gB proposed to be responsible for virus binding to host cells, fusion and cell-to-cell spread was the most highly conserved region with RhCMV gB (74% identity/85% similarity). Conserved elements included 11 of 12 cysteine residues, 14 of 16 potential N-linked glycosylation sites and cross-reactive epitopes. Metabolic labelling experiments demonstrated that RhCMV gB was proteolytically processed similarly to HCMV gB. These results are critical for investigating virus-host relationships in CMV-infected primates.