P-Rex1 is a specific guanine nucleotide exchange factor (GEF) for Rac, which is present in high abundance in brain and hematopoietic cells. P-Rex1 is dually regulated by phosphatidylinositol (3,4,5)-trisphosphate and the Gbetagamma subunits of heterotrimeric G proteins. We examined which of the multiple G protein alpha and betagamma subunits activate P-Rex1-mediated Rac guanine nucleotide exchange using pure, recombinant proteins reconstituted into synthetic lipid vesicles. AlF(-)(4) activated G(s),G(i),G(q),G(12), or G(13) alpha subunits were unable to activate P-Rex1. Gbetagamma dimers containing Gbeta(1-4) complexed with gamma(2) stimulated P-Rex1 activity with EC(50) values ranging from 10 to 20 nm. Gbeta(5)gamma(2) was not able to stimulate P-Rex1 GEF activity. Dimers containing the beta(1) subunit complexed with a panel of different Ggamma subunits varied in their ability to stimulate P-Rex1. The beta(1)gamma(3), beta(1)gamma(7), beta(1)gamma(10), and beta(1)gamma(13HA) dimers all activated P-Rex1 with EC(50) values ranging from 20 to 38 nm. Dimers composed of beta(1)gamma(12) had lower EC(50) values (approximately 112 nm). The farnesylated gamma(11) subunit is highly expressed in hematopoietic cells; surprisingly, dimers containing this subunit (beta(1)gamma(11)) were also less effective at activating P-Rex1. These findings suggest that the composition of the Gbetagamma dimer released by receptor activation may differentially activate P-Rex1.