contact sites is a critical event in the multistep process of cancer invasion. Recent studies indicate that the urokinase receptor (uPAR) is associated in large molecular complexes with other molecules, such as integrins. To test the possibility that uPAR may physically and functionally interact with vitronectin (Vn) receptors, we determined the expression level of uPAR, avb3, and avb5 Vn receptors in 10 human breast carcinomas. Here, we show the ability of uPAR to physically associate with avb5 in the breast carcinomas examined. The functional effects of this interaction were studied using HT1080 human fibrosarcoma and MCF-7 human breast carcinoma cell lines, both exhibiting a urokinase-dependent physical association between uPAR and avb5. Both cell lines respond to urokinase or to its noncatalytic amino-terminal fragment by exhibiting remarkable cytoskeletal rearrangements that are mediated by avb5 and require protein kinase C activity. On the contrary, binding of Vn to avb5 results in the protein kinase C-independent formation of F-actin containing microspiketype structures. Furthermore, avb5 is required for urokinase-directed, receptor-dependent MCF-7 and HT1080 cell migration. These data show that uPAR association with avb5 leads to a functional interaction of these receptors and suggest that uPAR directs cytoskeletal rearrangements and cell migration by altering avb5 signaling specificity.