The use of cryopreserved hepatocytes has increased in the last decade due to the improvement of the freezing and thawing methods, and has even achieved acceptance by the US Food and Drug Administration for use in drug metabolizing enzyme induction studies. This chapter provides an overview of the theories behind the process of cryopreservation as well as practical advice on methods to cryopreserve hepatocytes, which retain functions similar to fresh cells after thawing. Parameters, such as cell density, cryoprotectants, freezing media, storage conditions, and thawing techniques, should be critically considered. Special emphasis is put on human hepatocytes, but information for the cryopreservation of animal hepatocytes is also described.