Rat ascites hepatoma cells (MM I) invade a mesothelial cell monolayer in vitro in assay medium containing serum, but not in serum-free medium. Serum could be completely replaced by I -oleoyl lysophosphatidic acid (LPA) in inducing invasion. LPAinduced invasion was inhibited by genistein, a tyrosine-kinase inhibitor. Protein tyrosine phosphorylation in response to LPA was thus analyzed in order to determine the molecular mechanism of invasion. LPA of invasion-inducible concentrations evoked a transient increase in tyrosine phosphorylation, mainly of I IOto 130-kDa proteins in MMI cells but not in mesothelial cells. These concentrations of LPA were over I0 times higher (I 0 to 25 pM) than those necessary to produce a variety of biological actions, such as tyrosine phosphorylation in fibroblasts, neurite retraction and platelet aggregation. Protein tyrosine phosphorylation and invasion by MMI cells induced by LPA are largely regulated by rho p21, because both were inhibited by Clostridium botulinum C3 exo-enzyme, which is known to specifically inactivate rho p2 I. Invasion of MCL by MM I cells induced by serum and that by B l6FE7 cells induced by LPA were inhibited by genistein or C3 as well. By immunoprecipitation, we detected pl25 focal adhesion kinase (FAK) as a major protein of 110-to 130-kDa tyrosine phosphorylated in response to LPA. Tyrosine phosphorylation of paxillin by LPA was also detected.