Activation of phosphatidyl-inositol-3'-OH-kinase (PI3K) and the resulting production of phosphatidyl-inositol-3,4,5-trisphosphate (PIP3) are ubiquitous signaling steps that link various cell surface receptors to multiple intracellular targets. In fat and muscle cells, the same PI3K pathway that regulates metabolic enzymes, proliferation, and differentiation has also been shown to be involved in insulin-triggered insertion of glucose transporter GLUT4 into the plasma membrane. The multiple PI3K functions raise the question of how the same PI3K pathway can be selectively used for different cell functions. Here we developed a dual-color evanescent wave microscopy method to simultaneously measure PIP3 production and GLUT4 insertion in individual 3T3L1 adipocytes. Activation of PI3K was found to be both necessary and sufficient for triggering GLUT4 insertion, but transporter insertion was markedly suppressed for small-amplitude, persistent PIP3 signals and for large-amplitude, short PIP3 signals. The rejection of these common PI3K signaling responses may explain the selective advantage of insulin over platelet-derived growth factor and other stimuli for inducing GLUT4 insertion. Our study suggests that the same PI3K pathway can control specific cell functions by relying on effector systems that respond to particular receptor-encoded time courses and amplitudes of PIP3 signals.