We report that U373 glioblastoma cells constitutively producing human cytomegalovirus (HCMV) glycoprotein B (gB), the product of open reading frame UL55 of the HCMV genome, formed syncytia that contained 5 to 25 nuclei. Flow cytometry with a panel of monoclonal antibodies (mAbs) to the extracellular domain of HCMV gB showed that these cells expressed high densities of gB in the plasma membrane. We studied the properties of five clonal UB cell lines and the results are as follows. Reactivity of a panel of mAbs to HCMV gB showed that UB cells forming syncytia expressed gB with all of the conformational and sequential epitopes contained in the viral gB made in HCMV-infected cells. Syncytium formation in UB cells was independent of low pH and proteolytic cleavage of gB and was blocked by drugs that inhibit glycosylation and translocation of gB to the cell surface. Infected UB cells formed more syncytia than infected U373 cells, virions entered UB cells more rapidly, and higher virus titers were produced. Incubation of UB cells with complement-independent neutralizing antibodies to gB, which prevent virion entry into cells, cell-to-cell transmission of infection, and fusion of HCMV-infected glioblastoma cells, significantly reduced syncytium formation in UB cells. These results show that overlapping functional domains on HCMV gB promote fusion of the virion envelope with the cell surface, fusion of infected U373 cells, and syncytium formation in UB cell lines expressing high densities of gB in the plasma membrane. Our findings indicate that the functional regions of gB can be subjected to detailed analysis without constructing viral mutants by expressing mutated forms of gB with site-directed changes that preclude syncytium formation of U373 cells expressing these gene products.