Our goal is to understand the role of POPS, a lipid component only found in the inner leaflet of plasma membrane, in the organization of cholesterol in cell membranes. Our recent study of the transport behavior of cholesterol in POPS vesicles found that cholesterol transfer becomes significantly hampered at temperatures below 45C. Above 45C, the transport properties are similar to those of POPC. POPC and POPC are unsaturated lipids though POPS is anionic. The effect of arrested transport of cholesterol was observed with POPS's charge neutralized with NaCl. One explanation is the possible formation of cholesterol-rich regions within the POPS membrane. The goal of this proposal is to study the formation of cholesterol domains in POPS membranes as a function of temperature and POPC:cholesterol ratios and relate these to our observation of arrested cholesterol transport in POPS vesicles.