Although phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) is a well-characterized precursor for the second messengers inositol 1,4,5-trisphosphate, diacylglycerol  and phosphatidylinositol 3,4,5-trisphosphate , it also interacts with the actin-binding proteins profilin and gelsolin , as well as with many signaling molecules that contain pleckstrin homology (PH) domains . It is conceivable that stimuli received by receptors in the plasma membrane could be sufficiently strong to decrease the PtdIns(4,5)P2 concentration; this decrease could alter the structure of the cortical cytoskeleton and modulate the activity of signaling molecules that have PH domains. Here, we tested this hypothesis by using an in vivo fluorescent indicator for PtdIns(4,5)P2, by tagging the PH domain of phospholipase C delta 1 (PLC-delta 1) with the green fluorescent protein (GFP-PH). When expressed in cells, GFP-PH was found to be enriched at the plasma membrane. Binding studies in vitro and mutant analysis suggested that GFP-PH bound PtdIns(4,5)P2 selectively over other phosphatidylinositol lipids. Strikingly, receptor stimulation induced a transient dissociation of GFP-PH from the plasma membrane, suggesting that the concentration of PtdIns(4,5)P2 in the plasma membrane was effectively lowered. This transient dissociation was blocked by the PLC inhibitor U73122 but was not affected by the phosphoinositide (PI) 3-kinase inhibitor wortmannin, suggesting that it is mostly mediated by PLC and not by PI 3-kinase activation. Overall, our studies show that PtdIns(4,5)P2 can have second messenger functions of its own, by mediating a transient dissociation of proteins anchored in the plasma membrane.