Primary cultures of hepatocytes are useful tools for both short- and long-term pharmacotoxicological research. Under conventional conditions, isolated hepatocytes form a monolayer and survive for about 1 wk but lose some liver-specific functions, including xenobiotic biotransformation. In comparison with the conventional monolayer culture model, cocultures with rat liver epithelial cells (RLECs) have an extended life-span and better maintain their drug-metabolizing capacity, owing to the presence of cell-cell interactions. In this chapter, techniques for setting up conventional monolayer cultures and cocultures of hepatocytes with RLECs (including isolation, culture, and cryopreservation of RLECs) are described in detail. In addition, comments derived from our own experience are given for successfully culturing primary hepatocytes.