Endothelial migration on extracellular matrix is regulated by integrins and proteolysis. Previous studies showed that beta(3)-integrins regulate expression of the urokinase-type plasminogen activator receptor (uPAR) through outside-in signalling involving the cytoplasmic domain. Here we show that overexpression of the integrin-binding protein beta(3)-endonexin decreased uPAR promoter (-398 base-pair fragment) activity that is constitutively active in endothelial cells. Mutation of the NF-kappaB promoter binding site (-45 bp) impaired the ability of beta(3)-endonexin to downregulate uPAR promoter activity. Immunoprecipitation studies showed that beta(3)-endonexin interacts directly with the p50/p65 transactivation complex and thereby inhibits binding of kappaB oligonucleotides to the p50/p65 complex. Moreover, binding of beta(3)-endonexin to p50 was inhibited in the presence of kappaB but not mutated kappaB oligonucleotides, suggesting a sterical competition between beta(3)-endonexin and kappaB DNA for the p50/p65 complex. We therefore propose that beta(3)-endonexin acts as regulator of uPAR expression in beta(3)-integrin-mediated endothelial cell migration through direct interaction with p50/p65. Since NF-kappaB regulates the expression of matrix degrading enzymes, the present results define a role of beta(3)-endonexin in regulating beta(3)-integrin-mediated adhesion and pericellular proteolysis.