Understanding the primary steps of viral entry can have important implications for strategies to prevent infection of known viral pathogens as well as determining parameters for efficient gene delivery using viral vectors. Recently, a two-step process for viral infection involving attachment of virus to a primary receptor (coxsackievirus adenovirus receptor and heparan sulfate proteoglycan) and subsequent mediation of virus entry by a co-receptor (αV integrins and HVEM) has been determined for both adenovirus and HSV, respectively 1, 2, 3, 4. Heparan sulfate proteoglycan serves as a primary attachment receptor for adeno-associated virus type 2 (AAV-2)(ref. 5). Here we determined that αVβ5 integrin plays a part in efficient AAV infection. Experiments using the chelating agent EDTA to disrupt integrin function resulted in a corresponding decrease in AAV infection, consistent with the possibility that integrin mediates infection. Viral overlay experiments on purified plasma membrane proteins as well as immunoprecipitated integrin β5 subunit demonstrated that AAV directly associates with the β5 subunit of αVβ5 integrin. Genetically defined cells expressing αVβ5 integrin showed increased susceptibility to AAV infection, demonstrating a biological role of this integrin in AAV infection. Finally, viral binding and internalization studies indicate that αVβ5 integrin is not a primary attachment receptor for AAV-2, but is instead involved in facilitating virus internalization. This study supports the idea that αVβ5 integrin serves as a co-receptor for AAV-2 virions, and should have a substantial effect on the use of AAV vectors in human gene therapy.