We mapped three antigenic domains of continuous epitopes on human cytomegalovirus (CMV) glycoprotein B (gB) by reacting a panel of independently derived monoclonal antibodies with deletion mutants expressed transiently in COS-1 cells. One of these antigenic domains, DC2, maps in the last 75 amino acids of the carboxy terminus. These epitopes are conserved in strains Towne and AD169, as well as in 19 clinical CMV isolates. ELISAs of DC2-reactive antibodies with a set of overlapping synthetic oligopeptides from the carboxy terminus showed that the epitopes of antibodies CH405-1 and CH421-5 map between amino acids 833 and 852 and that the epitope of antibody CH28-2 maps between amino acids 878 and 898. These linear epitopes were grouped into domain DC3. The third antigenic domain, DC1v, maps at the amino-terminal end of CMV strain AD169 gB but is not contained in strain Towne or in 17 of 19 clinical isolates. Epitopes in this domain are likely to map between amino acids 28 and 67, an area where differences occur in the nucleotide sequence of the gB genes from AD169 and Towne. Analysis of CMV-infected cells by flow cytometry with antibodies to the amino- and carboxy-terminal domains revealed that the amino terminus of gB is extracellular and that the carboxy terminus is not exposed on the cell surface.