Parenchymal hepatocytes are responsible for most of the metabolic functions of the liver, but exhibit distinct functional properties depending on their localization within the hepatic lobule. Cytochrome P450 oxidases represent a family of drug-metabolizing enzymes, which are expressed predominantly in hepatocytes localized in the centrilobular area (zone 3). The present study describes a unique transgenic mouse strain that distinguishes zone 3 hepatocytes from periportal zone 1 hepatocytes by the intensity of EGFP fluorescence. Both zone 1 and zone 3 hepatocytes isolated from these mice showed the same zone-specific gene expression patterns as in liver tissue in vivo. Experiments using primary cultures of hepatocytes indicated that a combination of low oxygen concentration and activation of Wnt/b-catenin signaling maintained the expression of zone 3-specific P450 drug-metabolizing enzymes, which was characterized by their susceptibility to acetaminophen-induced mitochondrial dysfunction. These zonespecific hepatocytes provide a useful system in the research area of liver pathophysiology and drug development. (C) 2020 Elsevier Inc. All rights reserved.