INTRODUCTIONThe investigation of lipids in living cells is one of the underdeveloped areas in cell biology. Although it is possible to analyze the global lipid composition of a cell type, fractionation of the various types of membranes from cells is extraordinarily difficult, mainly because most membranes appear to be in contact with each other. Therefore, we know the lipid components, but we have a difficult time finding out their exact position, how dynamically they change location, and how rapidly they are metabolized. Imaging lipids in cells seems to be the obvious solution to the problem. The most common way to image molecules is by the artificial addition of a fluorescent tag. The use of fluorescent proteins has become the mainstay of protein imaging, but this method is, of course, not suitable for small molecules such as lipids. Unfortunately, the fluorescent tag is usually as large as the lipid and is therefore likely to have a severe influence on lipid location and metabolism. To circumvent this problem, two solutions have been developed--namely, the use of fluorescently labeled proteins that specifically recognize lipids and a chemical method to introduce the fluorescent tag inside the cell. This article describes procedures necessary to image lipids by fluorescently tagged lipid-binding domains and by labeling lipid derivatives in fixed and living cells.