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P-Rex1 is a guanine nucleotide exchange factor (GEF) for the small GTPase Rac that is primarily expressed in peripheral blood leukocytes and brain. P-Rex1 is directly and synergistically activated by both the phosphoinositide 3-kinase (P13K) product PtdIns(3,4,5)P3 and Gbg subunits of heterotrimeric G-proteins. Rac is a member of the Rho family of monomeric GTPases and is involved in a variety of cellular processes. P-Rex1 has been shown to provide a major link between P13K heterotrimeric G-proteins and Rac in neutrophils. P-Rex1 is an 185kDa protein containing a typical catalytic DH (or GEF) domain and tandem PH domain, two DEP and two PDZ domains and significant homology over its C-terminal half to Inositol Polyphosphate 4-Phosphatase. A panel of P-Rex1 deletion, truncation and point mutants have been created to investigate the mechanisms of activation of P-Rex1 Rac GEF activity by PtdIns(3,4,5)P3 and Gbg subunits. A P-Rex1 mutant lacking the PH domain (DPH) cannot be stimulated by PtdIns(3,4,5)P3 which implies that the PH domain meditates the activation of P-Rex1 Rac GEF activity by PtdIns(3,4,5)P3. Consistent with this, PtdIns(3,4,5)P3 binds to the PH domain of P-Rex1 and the isolated DHPH tandem is sufficient for PtdIns(3,4,5)P3-stimulated P-Rex1 Rac GEF activity. The Rac GEF activities of both the DPH mutant and the isolated DHPH tandem can be stimulated by Gbg subunits which implies that the catalytic DH domain mediates the activation of P-Rex1 Rac GEF activity by Gbg subunits. Deletion of the DEP, PDZ or the homology to Inositol Polyphosphate 4-Phosphatase domains has no major consequences on the abilities of either PtdIns(3,4,5)P3 or Gbg subunits to stimulate P-Rex1 Rac GEF activity. However, the presence of any of these domains impacts on the levels of basal and/or stimulated P-Rex1 Rac GEF activity, suggesting that there are important functional interactions between the DHPH tandem domains and the DEP, PDZ and homology to Ionsitol Polyphosphate 4-Phosphatase domains of P-Rex1.