We have constructed recombinant human adenovirus (Ad) vectors containing the glycoprotein gene of vesicular stomatitis virus (VSV). The structural gene of the VSV glycoprotein was modified by the addition of promoter and poly(A) addition sequences from the herpes simplex virus type 1 thymidine kinase (TK) gene and inserted, in either orientation, into early region 3 (E3) of human Ad type 5. The recombinant vectors were fully infectious and replicated in HeLa cells in culture. The TK promoter was functional in both insert orientations and responsive to trans-activation by herpes virus infection; however production of VSV glycoprotein in readily detectable amounts was only obtained with the vector having an insert in the E3 parallel orientation (AdG12), and depended principally on transcripts initiating within upstream Ad sequences. The onset of expression of the glycoprotein in AdG12-infected cells was detectable at about the same time as the Ad 72K DNA-binding protein encoded by E2, and its synthesis was not prevented by blocking viral DNA synthesis. The VSV glycoprotein produced by AdG12 was fully processed and could function to direct low pH-induced fusion of infected cells. These Ad vectors have considerable potential utility for the expression of antigens in cell culture and for the immunization of animals in studies of immunity and protection.