The surface potential of biological membranes varies according to their lipid composition. We devised genetically encoded probes to assess surface potential in intact cells. These probes revealed marked, localized alterations in the charge of the inner surface of the plasma membrane of macrophages during the course of phagocytosis. Hydrolysis of phosphoinositides and displacement of phosphatidylserine accounted for the change in surface potential at the phagosomal cup. Signaling molecules such as K-Ras, Rac1, and c-Src that are targeted to the membrane by electrostatic interactions were rapidly released from membrane subdomains where the surface charge was altered by lipid remodeling during phagocytosis.