Hepatocellular adenoma and focal nodular hyperplasia (FNH) are benign liver tumours. The differential diagnosis of these lesions and of well- to moderately differentiated hepatocellular carcinomas is often difficult but is very important in view of their different treatment. Although neither type of lesion is connected to the biliary tree, FNHs are cholestatic, whereas this is rarely the case for hepatocellular adenomas. This suggests that hepatocellular uptake and secretion of bile constituents is different in FNHs compared to adenomas. We therefore evaluated the expression and localization of hepatic transporters in hepatocellular adenomas, different types of FNH and well- to moderately differentiated hepatocellular carcinomas in non-cirrhotic liver and compared them with normal liver, using real-time RT-PCR and (semi-)quantitative immunohistochemistry. The parenchymal expression of the uptake transporter OATP2/8 (OATP1B1/3) was minimal or absent in adenoma, while there was strong and diffuse expression in FNH. We observed diffuse parenchymal expression of the basolateral export pump MRP3 in adenomas, while only reactive bile ductules and adjacent cholestatic hepatocytes were MRP3-positive in FNH. The MRP3/OATP2/8 expression pattern of atypical FNHs resembled that of adenomas, suggesting that both types of lesion are related. Most hepatocellular carcinomas showed decreased expression of one or more of the canalicular transporters (MDR1, MDR3, BSEP). The differences in transporter expression profile between FNHs and adenomas are most likely pathogenetically important and may explain why only FNHs are cholestatic. The finding that each type of focal lesion in non-cirrhotic liver has a specific transporter expression pattern may be useful in the establishment of a correct diagnosis by imaging or on needle biopsy.