The highly invasive human prostate cancer PC3 cell line was found to express the α_vβ₃ integrin; in contrast, the noninvasive LNCaP prostate cancer cell line did not express α_vβ₃. PC3 cells adhered to and migrated on vitronectin (VN), an α_vβ₃ ligand expressed in mature bone where prostate cancer cells preferentially metastasize. In contrast, LNCaP cells did not adhere to or migrate on VN. Analysis of primary human prostate cancer cells isolated from 16 surgical specimens, showed that these cells expressed α_vβ₃, whereas normal prostate epithelial cells did not. In addition, only primary prostate cancer cells adhered to and migrated on VN. The role of α_vβ₃ in mediating prostate epithelial cell migration was confirmed using LNCaP cell transfectants expressing β₃ (β₃-LNCaP). Exogenous expression of α_vβ₃ induced LNCaP cells to adhere to and migrate on VN. In response to α_vβ₃ engagement, increased tyrosine phosphorylation of focal adhesion kinase (FAK), a signaling molecule activated by integrins and able to modulate cell migration, was detected. Transfection of FAK-related nonkinase, known to compete with FAK for its correct localization and phosphorylation, caused inhibition of β₃-LNCaP cell migration, specifically on VN. These data indicate that de novo expression of α_vβ₃ integrin in prostate cancer cells generates a migratory phenotype that is modulated by a FAK signaling pathway. This study points to α_vβ₃ as potential target in prostate cancer cell invasion and metastasis.