Maturation of glycoprotein B (gB) of human cytomegalovirus (HCMV) includes a series of sequential glycosylation steps followed by proteolytic cleavage of the precursor protein. Inhibitors of glycosylation and glycoprotein processing, including tunicamycin, monensin, and bromoconduritol, were used to define further the processing pathway of HCMV gB. The results of these studies indicated that cotranslational glycosylation and intracellular transport are essential for subsequent cleavage of the precursor; early trimming in the endoplasmic reticulum is not a prerequisite but facilitates this processing event. Analysis of purified nuclei with gB-specific monoclonal antibody suggested that the mannoserich gB-precursor intermediate(s) is (are) compartmentalized in the nuclear fraction. Immunoelectron microscopy revealed that HCMV gB was localized in the outer as well as in the inner nuclear membranes of HCMV-infected fibroblasts. © 1990.