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Stable transfectants were selected from human astro-cytoma cells (U373) after transfection with recombinant expression vectors carrying the human cytomegalovirus (HCMV) glycoprotein B (gB; gpUL55) gene with alternative deletions of hydrophobic domain segment 1 (hdl) or segment 2 (hd2) of the carboxy-terminal potential bipartite membrane anchor domain. Com-parative analysis of HCMV gB forms from cell lines gB(Mhdl) and gB(Mhd2), expressing mutagenized gB, and those from ceils expressing authentic gB showed that deletion ofhdl, but not that ofhd2, interfered with efficient proteolytic leavage of the gB precursor. Both mutagenized gB forms exhibited correct transport to the cell surface. Deletion of hd2, but not that ofhdl, caused loss of membrane anchoring of the gB molecule, resulting in secretion of the respective gB form into the culture medium. The carboxy-terminal cleavage product of the soluble gB molecule, which migrated more slowly than its authentic counterpart, wasmodified by complex carbohydrate side chains and formed disulphide-linked complexes. Our observations indicate that hd2 is essential as well as sufficient for membrane anchoring of the HCMV gB molecule. For hdl, a potential fusogenic role is suggested by the conserved positional pattern of glycine residues, which is comparable to that of known fusion peptides of other viruses