Cholesterol localization in membranes, cells, and tissues is a topic of substantial and continuing interest to the biomedical cell biologist. Knowing precisely where and in what amount cholesterol is present offers potentially useful insights into the functional role of this lipid as a constituent of normal membranes, and the part it plays in the pathogenesis of diseases that involve dysfunction of cholesterol homeostasis. The cholesterol content of membranes influences a range of properties, such as fluidity, stability, the activities of membrane-bound enzymes, and the adhesive and electrical properties of the cell surface. Differences in cholesterol content characterize the various specialized membrane systems of the cell; plasma membranes and the membranes of endocytic organelles are normally cholesterol-rich, whereas endoplasmic reticulum, nuclear, and mitochondrial membranes typically have much lower cholesterol levels. Furthermore, the concept that cholesterol distribution may be heterogeneous within a given membrane, reflecting a mosaiclike pattern of functional differentiation, has also been widely debated. Despite the wealth of information from in vitro biophysical studies, however, there remain many gaps in our understanding of the functional significance of cholesterol distrtibution in relation to the diverse membrane-mediated cellular activities that are fundamental to issues function.