Cell migration is a fundamental aspect of the neoplastic cell metastasis. Here, we show that phosphatidylinositol (PI) 3-kinase is constitutively active and controls cell motility of highly invasive breast cancer cells by the activation of transcription factor, NF-kappaB. The urokinase-type plasminogen activator (uPA) promoter contains an NF-kappaB binding site, and uPA expression in MDA-MB-231 cells is induced by the constitutively active NF-kappaB. Thus, motility was inhibited by overexpression of a dominant negative p85alpha regulatory subunit of PI 3-kinase (p85DN), as well as by pretreatment of cells with specific inhibitors of the p110 catalytic subunit of PI 3-kinase, wortmannin and LY294002. The involvement of gene transcription in cell motility was suggested because treatment with actinomycin D and cycloheximide, which inhibit transcription and new protein synthesis, respectively, abolished endogenous migration of MDA-MB-231 cells. Although wortmannin, Ly294002, or overexpression of p85DN did not significantly reduce DNA binding activity of NF-kappaB in nuclear extracts, wortmannin, Ly294002, and the overexpression of p85DN or IkappaBalpha inhibited constitutive activation of NF-kappaB in a reporter gene assay. Highly invasive MDA-MB-231 cells constitutively secreted uPA in amounts significantly higher than poorly invasive MCF-7 cells. Furthermore, inhibition of NF-kappaB markedly attenuated endogenous migration, and inhibition of PI 3-kinase and NF-kappaB reduced secretion of uPA. Our data suggest a link between constitutively active PI 3-kinase, NF-kappaB, and secretion of uPA, which is responsible for the migration of highly invasive breast cancer cells. Thus, constitutively active PI 3-kinase controls cell motility by the regulation of expression of uPA through the activation of NF-kappaB.