Adult rat liver slices were maintained for 20 h in a novel organ culture system with minimal loss of viability and function. Potassium and adenosine triphosphate levels were maintained at in vivo levels, following an initial recovery period (2 to 4 h), for up to 20 h. Protein synthesis and secretion were linear for 20 and 16 h, respectively. In addition, the liver slices synthesized glycogen between 4 and 12 h in culture. Finally, the liver slices were hormonally responsive during the 20 h culture period as exemplified by glucagon-stimulated glucose production. This system provides a simple and effective method for the culture and biochemical maintenance of adult rat liver for 20 h with minimal loss of biochemical function.