Over the past several years, much progress has been made in the understanding of the molecular organization of phospholipid and protein in the erythrocyte membrane. However, very little is know about the localization of cholesterol, an important membrane constituent. This lack of information is due in part to the non reactivity of the cholesterol molecule. Thus the techniques which made possible the elucidation of the disposition of proteins and phospholipids in the membrane cannot be applied to the study of cholesterol. However, cholesterol exchanges freely between erythrocytes and plasma lipoproteins or sonicated lipid vesicles and furthermore, the cholesterol content of the membrane can be modified over a considerable range. In the study proposed here these features will be exploited to obtain information about the localization of cholesterol in the erythrocyte membrane and about the interaction of cholesterol movement between erythrocytes and vesicles will yield information about the distribution of cholesterol both in the plane of the membrane and across the membrane. The equilibrium distribution of cholesterol between erythrocyte membranes and sonicated phospholipid vesicles will be determined to obtain information about the relative affinities of cholesterol for different phospholipids. The equilibrium partition of cholesterol between ghosts and phospholipid vesicles containing erythrocyte transmembrane proteins will be studied to determine whether there is an interaction between cholesterol and these proteins. Experiments directed at elucidating mechanisms of cholesterol exchange between various membrane systems are proposed. A study of abnormal erythrocytes containing excess cholesterol (spur cells) will determine whether the excess cholesterol in these cells is segregated from the rest of the membrane cholesterol. The transmembrane distribution of cholesterol in these cells will be investigated using x-ray diffracton as well as kinetic techniques.