The liver is the largest internal organ in mammals, serving a wide spectrum of vital functions. Loss of liver function due to drug toxicity or viral infection is a major cause of death in the United States. The development of Bioartificial Liver (BAL) devices and the demand for pharmaceutical and cosmetic toxicity screening require the development of long-term hepatocyte culture techniques. However, primary hepatocytes rapidly lose their cuboidal morphology and liver-specific functions over a few days in culture. Accumulation of stress fibers, loss of metabolic function, and cell death are known phenomena. In recent years, several techniques were developed that can support high levels of liver-specific gene expression, metabolic and synthetic function for several weeks in culture. These include the collagen double-gel configuration, hepatocyte spheroids, coculture with endothelial cells, and micropatterned cocultures with 3T3-J2 fibroblasts. This chapter covers the current status of hepatocyte culture techniques, including: hepatocyte isolation, media formulation, oxygen supply, heterotypic cell–cell interactions, and basic functional assays.