Macrophage stimulating protein (MSP) is a ligand for the RON receptor protein tyrosine kinase. Activation of RON in murine resident macrophages results in cell shape change and migration. We studied cell movement induced by MSP in different types of human epithelial cells and the possible role of phosphatidylinositol-3 (PI-3) kinase in RON-mediated signal transduction. We observed specific and saturable binding of 125I-MSP to RON on several epithelial cell lines. In addition to activation and phosphorylation of RON, MSP also induced tyrosine phosphorylation of the PI-3 kinase p85 subunit in a time-dependent manner, with a peak at 15 min. Moreover, phosphorylated RON formed a complex with PI-3 kinase in both HK-NOC keratinocyte and RON cDNA-transfected MDCK cells. An in vitro protein interaction assay confirmed that PI-3 kinase from a lysate of MSP-activated cells bound to pure RON protein. MSP, at a concentration range of 1 to 5 nM, induced migration of three epithelial cell lines. This effect was inhibited by wortmannin, a specific inhibitor for PI-3 kinase, with an IC50 of 10 nM. MSP-induced shape change in murine resident peritoneal macrophages was also abolished by wortmannin. These data suggest that activation of PI-3 kinase is required for MSP-induced epithelial cell migration. The stimulation by MSP of epithelial cell movement may have implications for tissue repair, wound healing, and tumor metastasis.