Membrane ruffling and the closely linked response of fluid-phase pinocytosis were investigated in Chinese hamster ovary cells that stably overexpress the human insulin receptor and a mutant 85-kDa subunit of phosphoinositide (PI) 3-kinase (delta p85) that lacks a binding site for the catalytic 110-kDa subunit of this enzyme. Both membrane ruffling and pinocytosis induced by insulin were markedly impaired in these cells. Microinjection of Rac, a Ras-related small GTP-binding protein, induced membrane ruffling in human epidermoid carcinoma KB cells, and this effect of Rac was not blocked by coinjection of delta p85 or by exposure of cells to wortmannin, a specific PI 3-kinase inhibitor. These results suggest that PI 3-kinase is essential not only for insulin-stimulated membrane ruffling but also for pinocytosis, and that PI 3-kinase possibly functions upstream of Rac in the signal transduction pathway.