Rhesus cytomegalovirus (RhCMV) exhibits strong parallels with human CMV (HCMV) in terms of nucleic and amino acid identities, natural history, and mechanisms of persistence and pathogenesis in its natural host, rhesus macaques (Macaca mulatta). To determine whether this non-human primate model would be useful to assess vaccine strategies for HCMV, host immune responses to RhCMV glycoprotein B (gB) were evaluated in RhCMV-infected monkeys. Total protein extracts were prepared from cells transiently transfected with an expression plasmid for either the full-length gB or a derivative (gBDelta, 1-680 aa) lacking both the transmembrane domain and cytoplasmic tail. Western blot analysis showed identical reactivity of macaque sera with full-length gB and its derivative gBDelta, indicating that the immunodominant epitopes of gB are contained in the extracellular portion of the protein. Using gBDelta extract as a solid phase, a sensitive and specific ELISA was established to characterize gB antibody responses in monkeys acutely and chronically infected with RhCMV. During primary infection (seroconversion), gB-specific antibodies developed concurrently and in parallel with total RhCMV-specific antibodies. However, during chronic infection gB-specific antibody responses were variable. A strong correlation was observed between neutralizing and gB-specific antibody levels in RhCMV-seropositive monkeys. Taken together, the results of this study indicate that, similar to host humoral responses to HCMV gB, anti-gB antibodies are an integral part of humoral immunity to RhCMV infection and probably play an important protective role in limiting the extent of RhCMV infection. Thus, the rhesus macaque model of HCMV infection is relevant for testing gB-based immune therapies.