Murine monoclonal antibodies were used to identify an immunogenic envelope glycoprotein complex of guinea pig cytomegalovirus (GPCMV). The glycoprotein complex consisted of two virion components of estimated masses 60,000 and 90,000 Da. Murine monoclonal antibodies specific for the gp60-90 complex neutralized infectious virus in the presence of complement and detected expression of the envelope complex on the surface of infected cells. Pulse- chase and immunoblot analyses suggested that these virion forms were proteolytic products of an intracellular polyprotein precursor of an estimated molecular weight of 150,000 Da (gp150). In addition, the intracellular forms of this envelope glycoprotein complex existed as a disulfide-linked oligomer. The intracellular polyprotein was synthesized as a 100,000-Da precursor which was likely cotranslationally glycosylated using N- linked linkages to the major intracellular form, gp150. Because of the similarities in size, expression, and processing pathway, these results suggested that the GPCMV gp60-90 complex may represent the HCMV gB homolog of GPCMV. © 1994 Academic Press, Inc.