Preparations of isolated liver cells, either freshly prepared or in culture, have been available for many years; however, because they lack the polarization of the cell in the tissue, their application to the study of processes involved in bile formation has been very limited. The hepatocyte couplet model offers a unique opportunity to study in vitro the intracellular processes involved: not only the physiology and pathophysiology of bile formation, but also the corresponding structural and molecular disturbances underlying different experimental models of cholestasis. In this review we describe the experimental data generated from this model in our laboratory over a period of years, including application of hepatocyte couplets to the study of cytoskeletal changes within the hepatocyte and mechanisms of hepatoprotection in experimentally induced, clinically relevant models of intrahepatic cholestasis.