Heterotrimeric G proteins, G12 and G13, have been shown to transduce signals from G protein-coupled receptors to activate Rho GTPase in cells. Recently, we identified p115RhoGEF, one of the guanine nucleotide exchange factors (GEFs) for Rho, as a direct link between Galpha13 and Rho [Kozasa, T., et al. (1998) Science 280, 2109-2111; Hart, M. J., et al. (1998) Science 280, 2112-2114]. Activated Galpha13 stimulated the RhoGEF activity of p115 through interaction with the N-terminal RGS domain. However, Galpha12 could not activate Rho through p115, although it interacted with the RGS domain of p115. The biochemical mechanism from Galpha12 to Rho activation remained unknown. In this study, we analyzed the interaction of leukemia-associated RhoGEF (LARG), which also contains RGS domain, with Galpha12 and Galpha13. RGS domain of LARG demonstrated Galpha12- and Galpha13-specific GAP activity. LARG synergistically stimulated SRF activation by Galpha12 and Galpha13 in HeLa cells, and the SRF activation by Galpha12-LARG was further stimulated by coexpression of Tec tyrosine kinase. It was also found that LARG is phosphorylated on tyrosine by Tec. In reconstitution assays, the RhoGEF activity of nonphosphorylated LARG was stimulated by Galpha13 but not Galpha12. However, when LARG was phosphorylated by Tec, Galpha12 effectively stimulated the RhoGEF activity of LARG. These results demonstrate the biochemical mechanism of Rho activation through Galpha12 and that the regulation of RhoGEFs by heterotrimeric G proteins G1213 is further modulated by tyrosine phosphorylation.