We have previously shown that urokinase receptor physically and functionally interacts with αvβ5 vitronectin receptor, leading to tumor breast cell migration and invasion. Here, the link between these 2 receptors was further investigated by analyzing the expression levels of urokinase receptor and αvβ5 integrin in 35 human breast carcinomas and 5 benign breast lesions. The occurrence of a positive correlation between urokinase receptor and αvβ5 protein levels in benign and malignant tumor specimens prompted us to investigate whether engaged urokinase receptors might modulate αvβ5 expression. Here, we report the receptor‐dependent ability of catalytically inactive urokinase to upregulate the αv and β5 chains in MDA‐MB‐231 and MCF‐7 breast carcinoma cell lines in a time‐ and concentration‐dependent manner. This effect is dependent on protein kinase C activity and requires new protein synthesis. Accordingly, the availability of assembled αvβ5 receptors on the cell surface increases upon urokinase treatment, as shown by immunoprecipitation and immunocytochemical analyses. Exposure to urokinase leads to enhanced tumor cell migration and invasion, which is prevented by the “phosphorylation‐like” urokinase receptor antagonist His‐uPA^138E/303E, the DNA‐binding drug mithramycin, the protein kinase C inhibitor calphostin C and anti‐αvβ5 antibodies. Finally, urokinase enables benign breast MCF‐10A cells to cross Matrigel in a αvβ5‐ and urokinase receptor‐dependent manner, indicating that urokinase controls a regulatory circuitry crucial to breast tumor progression. © 2002 Wiley‐Liss, Inc.