Adenovirus contains a heterodimeric protein complex consisting of 188 kd fiber protein that mediates high affinity virus attachment to cells and a 400 kd pentavalent subunit (penton base) that contains five Arg-Gly-Asp sequences, implying a role for integrins in adenovirus infection. We demonstrate that the vitronectin-binding integrins a+ and a& promote viral infection in a novel way since antibodies against these receptors or soluble penton base block virus internalization without affecting attachment. Moreover, adenovirus binds to cultured cells lacking av integrins but fail to become internalized, thus restricting infection of these cells. Transfection of a,(-) cells with a cDNA encoding aV results in the expression of integrins a& and a& and allows virus internalization and infection. These data indicate that adenovirus attachment and uptake into cells are separate but cooperative events that result from the interaction of distinct viral coat proteins with a receptor for attachment and a, integrin receptors for internalization.