Rab5 is a small monomeric GTPase involved in regulating vesicle fusion events during receptor-mediated endocytosis. During endocytosis of the activated platelet-derived growth factor receptor, phosphatidylinositol 3-kinase (PI3K) remains associated with the receptor. We have found that the p85 alpha subunit of PI3K binds directly to Rab5 and possesses GTPase-activating protein (GAP) activity toward Rab5. We describe two methods used to characterize the GAP activity of p85 toward the Rab5 protein. The first method is a steady-state GAP assay, used to show that the p85 alpha protein has GAP activity toward Rab5. The second method is a single turnover GAP assay and measures changes in the catalytic rate of Rab5 GTP hydrolysis with or without the p85 alpha protein.