Bile secretion is an essential function of the liver and serves as a major route for the elimination and detoxification of many endogenous substances and xenobiotics, e.g. bile acids, bilirubin, cholesterol, phospholipids, and drugs. In addition, bile acids are required for the digestion and absorption of lipids from the intestinal lumen. Hepatobiliary elimination of metabolic end products and of drugs can be regarded as a vectorial process involving the uptake of substances from blood into the hepatocytes, frequently followed by their biotransformation by Phase I and Phase II metabolizing enzymes, and finally their efflux into bile. A broad range of transport proteins has been identified in the basolateral (sinusoidal) and apical (canalicular) membrane of hepatocytes that mediate the uptake and efflux of various biliary compounds, respectively, thus determining their clearance. The expression of many metabolizing enzymes and transport proteins is coordinately regulated by nuclear receptors that are activated by specific ligands, including numerous xenobiotics. The efflux of a large number of compounds into bile is driven by different ATP-binding cassette (ABC) transporters localized at the canalicular hepatocyte membrane. They include the bile salt export pump BSEP (ABCB11), the phospholipid flippase MDR3 (ABCB4), the apical conjugate efflux pump MRP2 (ABCC2), the breast cancer resistance protein BCRP (ABCG2), the long-known MDR1 P-glycoprotein (ABCB1), as well as the heterodimer of ABCG5 and ABCG8, which critically promotes biliary cholesterol secretion. BSEP mediates the ATP-dependent transport of taurine and glycine conjugates of bile acids across the canalicular membrane and is, thus, the main driving force of bile salt-dependent bile flow. MDR3 acts as a primary active phospholipid flippase and translocates phosphatidylcholine from the inner to the outer leaflet of the canalicular membrane. MRP2 accepts a broad range of anionic substrates, including substances conjugated with glutathione, glucuronate, or sulfate (e.g. leukotriene C4, bilirubin glucuronosides, and some steroid sulfates) as well as unconjugated anionic drugs. MRP2 contributes most significantly to the generation of bile salt-independent bile flow. BCRP mainly transports sulfated conjugates of steroids and xenobiotics. MDR1 generally does not accept anionic conjugates, but rather transports neutral and cationic organic compounds; however, some organic anions have been identified as substrates of MDR1 as well. The localization of several conjugate-transporting members of the ABCC subfamily at the sinusoidal membrane of hepatocytes, i.e. MRP3 (ABCC3), MRP4 (ABCC4), and MRP6 (ABCC6), indicates that organic anions and drug conjugates destined for renal elimination are also effluxed from hepatocytes into blood. Any functional disturbance of these transport systems can lead to the intracellular accumulation of potentially harmful xenobiotic and endogenous compounds and, therefore, constitutes a risk for the development of cholestatic liver disease. This is corroborated by the fact that mutations in ABC transporter genes resulting in functionally inactive canalicular transporters are the molecular basis of several inherited liver diseases. For instance, progressive familial intrahepatic cholestasis type 2 (PFIC2) and benign recurrent intrahepatic cholestasis type 2 (BRIC2) both represent hereditary BSEP deficiency syndromes. The hereditary absence of MDR3 expression leads to PFIC3, and lack of a functionally active MRP2 is the molecular basis of Dubin-Johnson syndrome, which is characterized by conjugated hyperbilirubinemia. Mutations abolishing the function of either the ABCG5 or the ABCG8 transporter result in sitosterolemia. Besides investigating genetic variants causing inherited liver disease, major research efforts are also undertaken to identify non disease-associated genetic polymorphisms of ABC transporters and to analyze their functional consequences. These transporter variants may account for interindividual variation, e.g. in multidrug resistance, pharmacokinetics of drugs, and adverse drug reactions. For instance, altered expression and function of MDR1 in human small intestine has been associated with changes in drug absorption, pointing toward a possible role of canalicular MDR1 expression levels in determining biliary elimination of drugs and hence hepatic exposure to xenobiotics. Recent studies demonstrated a considerable interindividual variability of canalicular ABC transporter expression in normal liver and a polymorphic transporter expression pattern, which might constitute a risk factor for the development of acquired forms of cholestatic liver diseases.