The gene of the human cytomegalovirus (HCMV) major envelope glycoprotein, gB, was cloned from the Towne strain and inserted into adenovirus type 5 downstream of the E3 promoter. The recombinant virus, Ad-gB, expressed antigenically related proteins of 58, 30, 25, and 23 kDa in A549 and MRC-5 cells; the 58-kDa protein had the same mobility as the native gB from HCMV-infected MRC-5 cells and virions. All four proteins were detected by a monospecific polyclonal antiserum and by a monoclonal antibody in immunoblot and immunofluorescence assays. Hamsters infected intranasally with live Ad-gB developed protein-specific and HCMV-neutralizing antibody. This study confirms the importance of gB in the generation of the neutralizing immune response to HCMV and demonstrates the potential of live adenoviruses as vaccine vectors.