Human cytomegalovirus (HCMV) glycoprotein B (gB) is an abundant glycoprotein in the virion envelope that elicits neutralizing antibodies in human infection and in immunized animals. Using a panel of monoclonal antibodies to gB with neutralizing activity, we analyzed in detail the mechanisms by which these antibodies block HCMV infection. Twelve antibodies with complement-independent neutralizing activity were studied for their effect on the attachment of virions to the cell surface, virion penetration into cells, the transmission of infection from cell to cell, and fusion of infected cells. All of the complement-independent antibodies blocked penetration of virions into cells but had no effect on virion attachment to the cell surface. The most potent neutralizing antibodies also limited the spread of infection from cell to cell. Fusion of HCMV-infected U373 glioblastoma cells was blocked by all but two of the neutralizing antibodies to gB, suggesting that fusion of infected cells is similar but not identical to the fusion of the virion envelope with the cell membrane that occurs during virion entry. Our findings provide the first evidence that HCMV gB is a multifunctional glycoprotein that promotes virion penetration into cells, the transmission of infection from cell to cell, and fusion of infected U373 cells.