The gene encoding glycoprotein B of human cytomegalovirus (CMV) strain Towne was cloned, sequenced, and expressed in order to study potential targets for viral neutralization. Secondary structure analysis of the 907 amino acid protein predicted a 24 amino acid N-terminal signal sequence and a potential transmembrane region composed of two domains, 34 and 21 amino acids. The CMV (Towne) gB gene had a 94% nucleotide similarity and a 95% amino acid similarity to the CMV (AD169) gB gene [as described by M.P. Cranage et al. (1986, EMBO J. 5, 3057-3063)]. Transcriptional analysis of the CMV (Towne) gB coding strand revealed that the gB message (3.9 kb), was transcribed from this region as early as 4 hr postinfection, and well in advance of gB protein synthesis. Full-length and truncated versions of the gB gene were expressed in COS cells using expression vectors where transcription was driven by the SV40 early promoter or the CMV major immediate early promoter. Expression was detected by immunofluorescence and ELISA using the virus neutralizing murine monoclonal antibody 15D8 (L. Rasmussen, J. Mullenax, R. Nelson, and T.C. Merigan, 1985, J. Virol. 55, 274-280). This antibody had been shown previously to recognize a 55-kDa CMV virion protein and a related 130-kDa intracellular precursor. Amino acid sequence analysis of the N-terminus of the 55-kDa viral glycoprotein (gp55) showed that gp55 is derived from gB (gp130) by proteolytic cleavage and represents the C-terminal region of gp130. The truncated version of gB expressed in COS and CHO cells was also processed by proteolytic cleavage as demonstrated by Western blotting. Our study localizes the epitope recognized by 15D8 to within a 186 amino acid fragment of the gp55 protein. These results indicate that CMV gB is a target for neutralization and establishes gp55 as a candidate component for use in a subunit vaccine.