Autoradiographic studies have shown that the liver accumulates endothelin. High-affinity binding sites for endothelin have been identified on rat liver plasma membranes. We investigated the role of endothelin isopeptides as mediators of cholestasis with isolated rat liver perfused by a recirculating solution of buffer and blood. These studies demonstrated that endothelin-1, as measured by means of radioimmunoassay, was cleared from the perfusate by the liver and that the liver concentrated both endothelin-1 and endothelin-3 in bile. Addition of endothelin-1 to the liver perfusate solution increased the concentration of endothelin-3 measured in the perfusate, suggesting that endothelin-1 caused release or secretion of endothelin-3. Both endothelin-1 and endothelin-3 at 5 nmol/L caused almost complete cessation of bile flow, but this effect was more prolonged after endothelin-1 than after endothelin-3 administration. Because it has been reported that cyclosporine increases endothelin levels, we studied the interaction of these two compounds. Cyclosporine (100 μmol/L) also produced cholestasis. Endothelin-3 secretion in bile, however, was decreased in livers perfused with cyclosporine compared with secretion in controls. Simultaneous addition of endothelin-1 and cyclosporine that on their own were not significantly cholestatic produced cholestasis. In conclusion, endothelin is a potent cholestatic agent secreted and excreted by the liver. It may potentiate the cholestatic action of cyclosporine. (HEPATOLOGY 1993;18:961-968.)