Cholesterol is an essential structural component in the cell membranes of most vertebrates. The biophysical properties of cholesterol and the enzymology of cholesterol metabolism provide the basis for how cells handle cholesterol and exchange it with one another. A tightly controlled — but only partially characterized — network of cellular signalling and lipid transfer systems orchestrates the functional compartmentalization of this lipid within and between organellar membranes. This largely dictates the exchange of cholesterol between tissues at the whole body level. Increased understanding of these processes and their integration at the organ systems level provides fundamental insights into the physiology of cholesterol trafficking.