Isolated hepatocytes obtained by collagenase perfusion of adult rat livers were seeded on collagen gels and kept in a chemically defined culture medium (except for the first 6 h of culture where 10% fetal calf serum was added). Cells adopted an epitheloid shape within 4 h and arranged themselves in a trabeculae-like pattern during the first 20 h of culture. In the electron microscope numerous tight junctions and bile capillaries were observed at sites of cell-to-cell contact. From metabolite analyses in the culture medium the following conclusions can be drawn: The cells continued to synthesize urea and ketone bodies for 5 days of culture. The cytosolic and mitochondrial redox states of the nicotinamide adenine nucleotide systsm were as in the liver in vivo and the oxygen supply of hepatocytes was sufficient under the culture conditions. Maximal velocities of ketogenesis from octanoate and of urea formation from ornithine plus ammonium chloride were stable during a 120 h culture period and compared well with rates found in the isolated perfused rat liver.