Human adenovirus type 2 (Ad2) enters host cells by receptor-mediated endocytosis, an event mediated by the virus penton base binding to cell surface integrins alpha v beta 3 and alpha v beta 5. While both alpha v integrins promote virus internalization, alpha v beta 5 is involved in the subsequent event of membrane permeabilization. Cells transfected with the beta 5 or beta 3 subunit, expressing either alpha v beta 5 and alpha v beta 3, respectively, were capable of supporting Ad2 infection to varying degrees. In this case, cells expressing alpha v beta 5 were significantly more susceptible to Ad2-induced membrane permeabilization, as well as to Ad2 infection, than cells expressing alpha v beta 3. Adenovirus-mediated gene delivery was also more efficient in cells expressing alpha v beta 5. These results suggest that the interaction of alpha v beta 5 with Ad2 penton base facilitates the subsequent step of virus penetration into the cell. These studies provide evidence for the involvement of a cellular receptor in virus-mediated membrane permeabilization and suggest a novel biological role for integrin alpha v beta 5 in the infectious pathway of a human adenovirus.