Herpes simplex virus type 1 (HSV-1) glycoprotein B (gB) is a major component of infected cell membranes and virion envelopes. Glycoprotein B is known to be essential for entry of viruses into cells and may play important roles in virus-induced cell fusion and other alterations in cell morphology. In order to study the biochemical and immunological properties of gB in isolation from other HSV-1 polypeptides we have constructed human adenovirus vectors capable of expressing high levels of gB. The gB gene was coupled to the SV40 early promoter and inserted into the E3 region of two adenovirus vectors, one in which the E1 region was deleted (AdgB-1) and another which contained E1 sequences (AdgB-2). In AdgB-1 the orientation of the chimeric gB-SV40 gene was right to left, i.e., opposite to the direction of late and E3 mRNA transcription, whereas in AdgB-2 the orientation was left to right. Human 293 cells which express E1 functions supported replication of AdgB-1 and gB was expressed in these cells but not in mouse cells and only at very low levels in human cells other than 293. Replication of AdgB-2 was not limited to 293 cells and the virus was able to induce synthesis of gB at levels equal to or higher than those expressed in HSV-1-infected human or mouse cells. Microscopic examination of AdgB-2-infected cells revealed extensive vacuolization in a manner completely uncharacteristic of adenovirus-infected cells, and fluorescent antibody staining indicated that gB was not only present at the cell surface but also concentrated in the cytoplasmic vacuoles.