We have previously shown that the transcellular migration of rat ascites hepatoma (AH130-MM1) cells through a cultured mesothelial cell monolayer (MCL) is triggered with lysophosphatidic acid (LPA) that stimulates actin polymerization and myosin light chain phosphorylation through the activation of Rho-ROCK (Rho-kinase) cascade. When, however, the motility of MM1 cells on a glass surface was tested by phagokinetic track motility assay, LPA failed to induce the motility. Nevertheless, when the glass had been coated with fibronectin (FN), LPA could induce phagokinetic motility which was accompanied by transformation of MM1 cells to fusiform-shape and assembly of focal adhesion. beta1 integrin, the counter receptor of FN, was expressed on MM1 cells. Anti-FN antibody, anti-beta1 integrin antibody and cyclo-GRGDSPA remarkably suppressed LPA-induced phagokinetic motility. These antibodies suppressed LPA-induced transcellular migration through MCL, as well. These results indicate that actin polymerization and phosphorylation of myosin light chain through Rho activation are insufficient for inducing motility but the cooperative FN/beta1 integrin-mediated adhesion is necessary for both the phagokinetic motility and transcellular migration of MM1 cells.