Rab5 and Rab4 are small monomeric GTPases localized on early endosomes and function in vesicle fusion events. These Rab proteins regulate the endocytosis and recycling or degradation of activated receptor tyrosine kinases such as the platelet-derived growth factor receptor (PDGFR). The p85␣ subunit of phosphatidylinositol 3-kinase contains a BH domain with sequence homology to GTPase activating proteins (GAPs), but has not previously been shown to possess GAP activity. In this report, we demonstrate that p85␣ has GAP activity toward Rab5, Rab4, Cdc42, Rac1 and to a lesser extent Rab6, with little GAP activity toward Rab11. Purified recombinant Rab5 and p85␣ can bind directly to each other and not surprisingly, the p85␣-encoded GAP activity is present in the BH domain. Because p85␣ stays bound to the PDGFR during receptor endocytosis, p85␣ will also be localized to the same early endosomal compartment as Rab5 and Rab4. Taken together, the physical co-localization and the ability of p85␣ to preferentially stimulate the down-regulation of Rab5 and Rab4 GTPases suggests that p85␣ regulates how long Rab5 and Rab4 remain in their GTP-bound active state. Cells expressing BH domain mutants of p85 show a reduced rate of PDGFR degradation as compared with wild type p85 expressing cells. These cells also show sustained activation of the mitogen-activated protein kinase and Akt pathways. Thus, the p85␣ protein may play a role in the down-regulation of activated receptors through its temporal control of the GTPase cycles of Rab5 and Rab4.Down-regulation of signal transduction pathways activated by receptor tyrosine kinases, such as the PDGFR, 1 includes endocytosis of the activated receptor complex (1, 2). Receptormediated endocytosis involves multiple vesicle fusion events that effectively deliver the receptor-signaling complex to the early endosome. This complex is then disassembled and the receptor is either recycled back to the plasma membrane or sorted to the late endosome and lysosome for degradation (3, 4). Rab5 is a small monomeric GTPase involved in early endosomal fusion events such as the fusion of clathrin-coated vesicles (containing activated receptors undergoing endocytosis) with the early/sorting endosomes (reviewed in Refs. 5 and 6). GDP-bound Rab5 is inactive and bound to a guanine dissociation inhibitor (GDI) protein in the cytosol. GTP-bound Rab5 is active and localized on the cytoplasmic face of early/sorting endosomes where it is involved in binding specific effector proteins such as the early-endosomal autoantigen 1 (EEA1) (7,8). EEA1 is a cytosolic protein that is recruited to early endosomal membranes by binding to Rab5-GTP and the lipid product of the class III PI3K p150/hVPS34, phosphatidylinositol 3Ј-phosphate (7, 9, 10). EEA1 is a core component required for early endosomal fusion events (7,8,11,12). The half-life of phosphatidylinositol 3Ј-phosphate, and the duration of Rab5-GTP have been suggested to influence the rate and extent of the endosome fusion reaction (13). The low intrinsic GTPase act...