SUMMARY Phospholipid flip-flop on cell membranes can exert profound impacts on cellular signaling and functions, including apoptosis, phagocytosis, blood coagulation, membrane vesicle shedding, bone mineralization, cell-cell fusion, fertilization, viral infection including HIV and SARS-CoV2 infections. Nevertheless, how phospholipid flip-flop leads to the observed cellular responses is largely elusive. The recent identifications of phospholipid scramblases and flippases have enabled genetic manipulations of these critical phospholipid transporters, which greatly advanced our understanding on the biology of phospholipid flip-flop. However, phospholipid flip-flop is a dynamic process and the genetic manipulations only allow us to observe the end results, which hinders gaining mechanistic understanding phospholipid flip-flop in various physiological process in real time. In this application, we aim to test the feasibility of developing a genetically encoded, optogenetic toolbox to precisely control phospholipid flip-flop with light at high temporal and spatial resolution and in real time. Our proposal is based on our extensive experience on the recently discovered calcium-activated phospholipid scramblase (CaPLSase) TMEM16F at molecular and cellular levels. In response to intracellular calcium increase, TMEM16F can rapidly catalyze phospholipid flip-flop, efficiently disrupt membrane environment and trigger wide spectrum of cellular changes. Here, we will use two complementary but independent approaches to develop the optogenetic tools to control phospholipid flip-flop. First, we will coexpress various calcium- mobilizing optogenetic tools to indirectly activate TMEM16F utilizing its calcium sensing property. Second, we will engineer light sensing motifs into TMEM16F to enable direct light control of its activities. If successful, the optogenetic toolbox developed in this high-risk, high-reward application will have profound impacts and broad applications in membrane biology, cell biology, physiology, hematology, immunology, virology and medicine.