We examined whether constitutively active mutants of the Gα proteins Gα12and Gα13, which together comprise the G12subfamily of Gα proteins, induce Rho-dependent tyrosine phosphorylation of the focal adhesion proteins p125 focal adhesion kinase, paxillin, and p130 Crk-associated substrate. We report that transient expression of the constitutively active mutants of Gα12or of Gα13in human embryonic kidney 293 cells stimulates tyrosine phosphorylation of a set of proteins ofMrof 110,000–130,000, 97,000, and 60,000–70,000. We identified p125 focal adhesion kinase, paxillin, and p130 Crk-associated substrate as prominent tyrosine-phosphorylated proteins in human embryonic kidney 293 cells expressing constitutively active Gα12and Gα13. In common with the increased tyrosine phosphorylation of these proteins mediated by mitogens acting through heptahelical receptors, the Gα12- and Gα13-mediated increase in tyrosine phosphorylation is blocked by cytochalasin D, which specifically disrupts the actin cytoskeleton, and by the Clostridium botulinumC3 exoenzyme, which ADP-ribosylates and inactivates Rho. Our results support the hypothesis that Gα12and Gα13activate Rho and suggest that Gα12and Gα13may mediate the tyrosine phosphorylation of p125 focal adhesion kinase, paxillin, and p130 Crk-associated substrate.