Hepatocyte couplets, by retaining functional and morphological polarity similar to that of hepatocytes in situ, are a valuable in vitro model to study mechanisms of bile secretion, cholestasis and hepatocellular injury. They have been useful in studies of the hormonal control of bile formation and are suitable for morphological studies. The availability of periportal- and perivenous-enriched couplet populations now allows a zonal perspective. Their contribution to our understanding of regulatory aspects of hepatobiliary dysfunction due to toxicological or cholestatic insult, as well as its reversion by using hepatoprotective agents, is still at an early stage. The next few years should see further exiting contributions to our understanding of hepatobiliary function and dysfunction.