Botulinum C3 exoenzyme was used to specifically ADP-ribosylate and inactivate rho p21, and the effects of rho p21 inactivation on lysophosphatidic acid (LPA)-induced tyrosine phosphorylation were examined in cultured Swiss 3T3 cells. LPA induced a rapid increase in the tyrosine phosphorylation of a number of proteins. Pretreatment of the cells with the C3 exoenzyme caused ADP-ribosylation of rho p21 in the cells and selectively attenuated the phosphorylation of several proteins, including p43 mitogen-activated protein kinase, p125 focal adhesion kinase, and two proteins of 72 and 88 kDa. C3 exoenzyme pretreatment did not block the initial phosphorylation and activation of mitogen-activated protein kinase but suppressed its subsequent rise. In contrast, the enzyme treatment inhibited the induction of phosphorylation of the 72- and 88-kDa proteins and suppressed the basal and LPA-induced tyrosine phosphorylation of p125 focal adhesion kinase. In addition, immunoprecipitation of cell lysates with an antibody directed against the 85-kDa subunit of phosphatidylinositol 3-kinase (PI 3-kinase) co-precipitated a tyrosine-phosphorylated band of 180 kDa. C3 exoenzyme pretreatment suppressed both the phosphorylation of this band and PI 3-kinase activation associated with LPA stimulation. These findings suggest that rho p21 works as a link between the LPA receptor signal and the subsequent tyrosine phosphorylation and PI 3-kinase activation in these cells.