Contortrostatin is a homodimeric disintegrin from snake venom. We have shown that contortrostatin binds to integrins alpha IIb beta 3, alpha 5 beta 1, and alpha v beta 3. We now use several criteria to demonstrate the binding of contortrostatin to alpha v beta 5. First, incubation of T24 cells, which express alpha v beta 3 and alpha v beta 1, with antibody against alpha v beta 3 failed to completely inhibit adhesion of cells to vitronectin, However, pretreatment of the cells with contortrostatin or the combination of antibodies against alpha v beta 3 and alpha v beta 3 completely blocked adhesion to vitronectin. By contrast, either anti-alpha v beta 5 alone or contortrostatin blocked adhesion of an alpha v beta 3-negative T24 subline, Second, contortrostatin as well as anti-alpha v beta 5 inhibits invasion of OVCAR-5, which express only alpha v beta 5. Third, contortrostatin binds to purified alpha v beta 5 in a saturable manner, Finally, radioligand binding assays yielded a K-d, value of 24 nM for [I-125]contortrostatin binding to alpha v beta 5. This investigation identifies alpha v beta 5 as a binding site for contortrostatin, Blockage of alpha v beta 5 by contortrostatin inhibits alpha v beta 5-mediated adhesion and invasion.